VCE Biology Unit 3 AOS 1: Nucleic Acids and Proteins (PDF)

Unit 3 AOS 1 - WHAT IS THE ROLE OF NUCLEIC ACIDS AND PROTEINS IN MAINTAINING LIFE? [CHAPTER 1] Dot point 1 - nucleic acids as information molecules that encode instructions for the synthesis of proteins: the structure of DNA, the three main forms of RNA (mRNA, rRNA and tRNA) and a co...

Unit 3 AOS 1 - WHAT IS THE ROLE OF NUCLEIC ACIDS AND PROTEINS IN MAINTAINING LIFE? [CHAPTER 1] Dot point 1 - nucleic acids as information molecules that encode instructions for the synthesis of proteins: the structure of DNA, the three main forms of RNA (mRNA, rRNA and tRNA) and a comparison of their respective nucleotides Nucleic acid DNA RNA Sugar Deoxyribonucleic acid Ribonucleic acid Nitrogen-containing bases Adenine, cytosine, guanine and thymine Adenine, cytosine, guanine and uracil No. of strands 2 (double stranded) 1 (single stranded) Nucleic acids 🡪 biomolecules vital for the continuity of life, in all organisms, provide genetic blueprint that provides the information for protein synthesis. Thymine / Uracil pairs with Adenine = 2 hydrogen bonds Cytosine pairs with Guanine = 3 hydrogen bonds Adenine and Guanine = purine Cytosine and Thymine / Uracil = pyrimidines All nucleic acids are made up of subunits called nucleotides which contains a phosphate, 5-carbon (pentose) sugar and a nitrogenous base DNA - DEOXYRIBONUCLEIC ACID DNA is vital code for all proteins 2 complementary chains of nucleotides Run anti-parallel (in opposite directions) One strand runs 3’ to 5’ and the opposite strand runs 5’ to 3’ The 5’ end is the phosphate end, which is attached to the 5’ carbon of the sugar. The 3’ end is the hydroxyl end of the sugar, which is associated with the 3’ carbon RNA - RIBONUCLEIC ACID Formed against a template of DNA Runs 5’ to 3’ Contains the base Uracil instead of Thymine 3 forms of RNA Messenger RNA (mRNA) 🡪 form of RNA synthesised by the transcription of a DNA template strand in the nucleus; mRNA carries a copy of the genetic information into the cytoplasm o Which carries the genetic message from the DNA within the nucleus to the ribosomes, where the message is translated into a particular protein. Each group of three nucleotides in mRNA (known as  a codon) provides the information for the addition of one amino acid. A special form of mRNA known as pre-mRNA is made through transcription in the nucleus. Ribosomal RNA (rRNA) 🡪 stable form of RNA found in ribosomes o rRNA combine w/ proteins and enzymes in the cytoplasm to form ribosomes o Ribosomes are where protein synthesis occurs Transfer RNA (tRNA) 🡪 form of RNA that can attach to specific amino acids and carry them to a ribosome during translation o molecules that carry amino acids to ribosomes that are free in the cytoplasm, where they are used to construct proteins. An anticodon (a set of three nucleotides) binds to the complementary codon on mRNA DOT POINT 2 - the genetic code as a universal triplet code that is degenerate and the steps in gene expression, including transcription, RNA processing in eukaryotic cells and translation by ribosomes Genetic code 🡪 representation of genetic information through a non-overlapping series of groups of three bases (triplets) in a DNA template chain one genetic instruction consists of a group of three bases (like AAT or GCT) = code is referred to as a triplet code The code is essentially the same in bacteria, in plants and in animals — it is said to be universal ○ same sequence of nucleotides codes for the same amino acid  the genetic code is that it is degenerate or redundant Degenerate 🡪 the property of the genetic code in which more than one triplet of bases can code for one amino acid ○ Code being degenerate it allows us to be more tolerant to mutations Unambiguous🡪 each codon is only capable of coding for a single amino acid STEPS IN GENE EXPRESSION TRANSCRIPTION process of copying the genetic instructions present in DNA to messenger RNA Location = nucleus It is important to remember that in mRNA, there is no thymine (T). This is replaced with uracil. STEPS (initiation, elongation, and termination) 1. RNA polymerase binds to the promoter sequence of DNA causing the double stranded DNA to unwind and unzip, exposing the bases of each strand 2. RNA polymerase catalyses transcription through the joining of complementary nucleotides onto the growing 3’ end of the mRNA strands 3. After the RNA polymerase moves to the downstream region of the gene, transcription stops, and the pre-mRNA which is complementary to the DNA template strand is released from the template. RNA PROCESSING occurs after transcription and involves modifying pre-mRNA to form mature mRNA; also known as post-transcription modification Introns are spliced out and exons are joined together to create mature mRNA Introns 🡪 parts of the coding region of a gene that are transcribed but not translated Exons 🡪 parts of the coding region of a gene that are both transcribed and translated Spliceosomes 🡪 complex molecules present in the nucleus that remove introns from the pre-mRNA transcript STEPS 1. Capping. 5’ end of pre-mRNA is capped with altered guanine base. Methyl cap protects pre-mRNA from the enzyme attack☹. Methyl cap helps pre-mRNA maintain stability, helping it attach to the ribosome 2. Adding a tail. Primary transcript is clipped at a specific point downstream of coding region and a poly-A- tail, with 250 As, is then added at the 3’ end. The poly A tail helps maintain stability of mRNA and simplifies export from the nucleus. 3. Splicing. Regions of pre-mRNA that contain introns are spliced out and remaining exons are joined together. Cutting and splicing is done by SPLICESOMES, which recognise specific base sequences at the end of the introns. GU at 5’ end and AG at 3’ end. 4. The mature mRNA now moves across the nuclear membrane into the cytosol, carrying with it a copy of the information originally encoded in the DNA of the genes ALTERNATIVE SPLICING one gene can be regulated in different ways so that it can produce more than one protein This occurs through techniques such as alternative splicing and exon juggling, leading to the production of different mRNAs and proteins from the same gene TRANSLATION process of decoding the genetic instructions in mRNA into a protein (polypeptide chain) built of amino acids The decoding of the genetic instructions Location = cytoplasm End of this process 🡪 genetic instructions carried in mRNA have been decoded and translated into a protein chain built of amino acids STEPS 1. Ribosome binds to and reads the mature mRNA in groups of 3 known as codons 2. tRNA, which carry amino acids, have anticodons which are complementary to the mRNA codons and are taken to the ribosome 3. The ribosome continues to move along the mRNA strand and as amino acids are delivered by tRNA molecules, they are joined together by peptide bons, a product of condensation reaction, forming a polypeptide chain DOT POINT 3 - The structure of genes: exons, introns and promoter and operator regions Coding region 🡪 part of a gene that contains the coded information for making a polypeptide chain Flanking regions 🡪 regions located either downstream or upstream of the coding region of a gene  Genes contain coding and non-coding sequences of bases Upstream region of gene contains base sequences that regulate its activity Contains promoter region sequence that allows for recognition and binding of RNA polymerase The termination sequence represents a sequence of DNA that signals for the end of transcription INTRONS AND EXONS Coding region part of a gene that contains the coded information for making a polypeptide chain Made up of introns and exons Exons contain the instructions for the synthesis of the protein and are both transcribed and translated. They provide the instructions that code for the amino acids in the produced protein. Introns are lengths of DNA that do not contain instructions relating to the protein chain. ○ Cut out during RNA processing, which means they are transcribed but not translated PROMOTER REGION - UPSTREAM part of the upstream flanking region of a gene where RNA polymerase binds that contains base sequences that control the activity of that gene TATA box 🡪 short base sequence consistently found in the upstream flanking region of the coding region of genes of many different species Without a functioning promoter region, transcription cannot be properly initiated LEADER REGION plays a critical role in one mode of regulating gene expression in prokaryotes In prokaryotes, it tends to be small, but can be much longer in eukaryotes upstream of the coding region, and downstream of the promoter and operator Contain sections known as attenuators and as such are involved in a process of gene regulation called attenuation OPERATOR REGION serves as the binding site for repressor proteins, which can then inhibit gene expression only found in prokaryotic genes  repressor protein a protein coded for by a regulatory gene that prevents gene expression by binding to its operator ○ prevents the RNA polymerase binding to the promoter, and thus transcription cannot be initiated bound with a repressor protein – RNA polymerase cannot move downstream from the promoter region, inhibiting transcription of the gene not bound with a repressor protein – RNA polymerase is free to move downstream from the promoter region, allowing for the transcription of the gene as usual DOT POINT 4 - The basic elements of gene regulation: prokaryotic trp operon as a simplified example of the regulatory process gene regulation the control of gene expression, typically achieved by switching transcription on or off Gene regulation involves the process of either inhibiting or activating gene expression ○ gene expression the process of reading the information stored within a gene to create a functional product, typically a protein Structural genes 🡪 genes that produce proteins that contribute to the structure or functioning of an organism responsible for producing proteins that are involved in the structure or function of a cell e.g ○ coding for enzymes, transport proteins, receptors, or peptide hormones. These genes are often found downstream (towards the 3’ end of the coding strand) of the regulatory gene that controls them Regulator genes 🡪 genes that produce proteins that control the activity of other genes Helps organisms conserve energy and ensures that cells produce the appropriate proteins TRP OPERON Trp operon 🡪 a series of genes within certain species of bacteria that encode for the production of the amino acid tryptophan An operon is group of linked structural genes with a common promoter and operator that is transcribed as a single unit Composed of: ○ Structural genes: a gene that codes for any RNA or protein product other than a regulator (trpA, trpB, trpC, trpD, and trpE, which encodes tryptophan synthetase) ○ A regulatory gene: a gene that codes for a product (typically protein) that controls the expression of other genes (usually at the level of transcription). In this case, the regulatory gene encodes for a repressor, which when active, binds to DNA and thus regulates the expression of genes by decreasing the rate of transcription. ○ A promoter (Ptrp): The promoter is a short DNA segment where RNA polymerase can attach and start transcription of the structural genes. The trp genes are transcribed as a single entity with one long mRNA transcript being produced. ○ An operator (O): An operator is a short DNA segment that provides a binding site for a repressor, so transcription cannot occur.  ○ A leader which contains the trpL gene (for the leader peptide) and attenuator section, that is able to further regulate transcription. ATTENUATION Low levels of tryptophan (or absence) 1. As tryptophan is absent (or low), trpL is translated slowly. As the ribosome translates the gene, the ribosome pauses at the trp codon to wait for enough tryptophan to be available to produce the leader polypeptide. 2. Due to this, the ribosome stays attached to region 1, and therefore no hairpin loop can form between the mRNA in regions 1 and 2 (as the ribosome is shielding the region). 3. Instead, a hairpin loop forms between the mRNA of regions 2 and 3. 4. This hairpin loop does not cause the RNA polymerase to detach (and thus is sometimes known as the antiterminator hairpin) and prevents the terminator hairpin of regions 3 and 4 forming. Therefore, RNA polymerase remains attached to the trp operon. 5. Transcription (and translation) of the trp operon continues and the proteins from the structural genes are produced. High levels of tryptophan (or presence) 1. As tryptophan is present, the ribosome does not stop at the two trp codon and trpL can be quickly translated. When the ribosome translates this region quickly (to the leader peptide), the ribosome quickly detaches from the mRNA. 2. This allows a hairpin loop to form between the mRNA produced in regions 1 and 2. 3. A terminator hairpin loop between the mRNA in regions 3 and 4 is therefore also able to form. 4. This terminator hairpin loops causes the RNA  polymerase to detach from the DNA. Transcription is stopped and the structural genes in the trp operon are not transcribed or translated. REPRESSION Presence of trp When tryptophan is present it binds to the repressor protein causing a configurational change allowing it to be active The active repressor protein can now bind to at the operator which means the RNA polymerase cannot bind at the operator and transcription does not occur OPERON IS OFF Absence of trp When tryptophan is not present the repressor is unable to bind to the operator (as it is still in an inactive form). The RNA polymerase is now able to the promoter and start transcription of the structural genes OPERON IS ON structural genes in the trp operon are only expressed when tryptophan is absent Describe the process of attenuation in the trp operon when tryptophan levels are high within a cell. Transcription and translation occur simultaneously. When tryptophan levels are high, the ribosome does not pause at the attenuator sequence which causes the mRNA molecule to fold into a terminator hairpin. This terminator hairpin folding causes the mRNA molecule to separate from the template DNA ending translation. Consequently, RNA polymerase detaches from the DNA ending transcription and no new tryptophan is produced. DOT POINT 5 - Amino acids as the monomers of a polypeptide chain and the resultant hierarchical levels of structure that give rise to a functional protein. Proteins are buildings block for life! They have complex structures Made from polypeptides Polypeptides 🡪 chains of covalently bonded amino acids STRUCTURE OF AMINO ACIDS each amino acid consists of a central carbon atom that is bonded to a hydrogen atom, a carboxyl group (COOH), an amino group (NH2 ), and an R-group the R-group uniquely determines the identity of a particular amino acid (varies)  each R-group has its own chemical properties, which can affect how different amino acids within a protein interact with each other ○ eg an amino acid with a hydrophobic R-group is more likely to form bonds with other hydrophobic R-group amino acids than it would with an amino acid containing a hydrophilic R-group Amino acids joined together by peptide bonds are called polypeptides ○ polypeptide a type of protein structure where three different folds of alpha helices, beta-pleated sheets and random coils can occur in amino acid chains, depending on the R groups in the different amino acids (long chain of amino acids) The joining of amino acids together occurs at a cell’s ribosomes via a condensation reaction, which results in the formation of peptide bonds between adjacent amino acid (requires ATP) ○ condensation reaction a reaction where two monomers join to form a larger molecule, producing water as a by-product ○ peptide bond the chemical bond linking two amino acids The hydroxyl group is broken off the carboxyl of one amino acid and forms a covalent bond with a hydrogen from another amino acid PROTEIN STRUCTURE Structure Description Image Primary - Linear sequence of amino acids in a polypeptide chain - Amino acids joined by peptide bonds - brought together as a polypeptide during translation in the ribosome Secondary - formed when a polypeptide chain folds and coils by forming hydrogen bonds between amino acids of its different sections - Folds happen depending on R group of amino acid - alpha (α) helices: coiled - beta-pleated (β) sheets: folded - random coils: irregular portions of secondary structure that join alpha-helices and beta-pleated sheets Tertiary - overall functional 3D shape of a protein - depends on both the primary and the secondary structures - The 3-D shape is critical for its function, where if the shae such as an active site is changed it would no longer function as an enzyme - minimum level of structure required to for a protein to be functional - formed when the secondary structures further fold by forming interactions and bonds between amino acids and R-groups of its different sections - i.e disulphide bonds Quaternary - Describes a structure in which two or more polypeptide chains with tertiary structure interact to form a protein - not all proteins will have a quaternary structure - Polypeptide chains with tertiary or quaternary structure can have a prosthetic group attached - prosthetic group a non-protein group bound to a protein. For example, a vitamin or ion - An example is haemaglobin Depending on the sequence of amino acids, the R-groups will interact with each other differently, forming different bonds that favour folding into specific 3D structures functional diversity of proteins arises due to the ability to create an unlimited number of complex combinations of amino acids that fold into polypeptides of varying shapes and sizes DOT POINT 6 - Proteins as a diverse group of molecules that collectively make an organism’s proteome, including enzymes as catalysts in biochemical pathways Proteins, also known as polypeptides, are one of the four types of biomacromolecules Are large complex structures which are crucial to the functioning and development of all living organisms, serving a variety of different functions enzyme an organic molecule, typically a protein, that catalyses (speeds up) specific reactions Function Description Enzyme - organic catalysts that speed up chemical reactions - e.g RNA polymerase: catalyses the formation of mRNA from DNA Transport - typically embedded in membranes, controlling the entry and exit of substances from a cell - e.g chloride channels, glucose channels, sodium-potassium pumps Structural - support cell and tissue shape - e.g collagen: found in connective tissues such as tendons and ligaments Hormones - many peptide hormones are chemical messengers used to communicate and induce changes in cells - peptide hormone a protein signalling molecule that regulates physiology or behaviour  - e.g insulin: regulates blood sugar levels Defence - involved in the immune system by recognising and destroying pathogens - e.g antibodies Motor/contractile - involved in the contraction and movement of muscles, the movement of internal cell contents around the cytoplasm, and the movement of cilia and flagella - e.g myosin and actin: work together to enable muscle contraction basic function of enzymes is to increase the rate of almost all the chemical reactions in living organisms and to do this within the prevailing conditions of temperature and pH within cells Enzymes are molecules that are organic (carbon-based) catalysts ○ catalyst a substance capable of increasing the rate of a reaction without being used up Most enzyme-catalysed reactions are reversible, with the same enzyme often capable of building up larger molecules (anabolic), or breaking them down into smaller ones (catabolic) Enzymes are reusable as they are not used up in the reaction an enzyme’s active site and substrate are complementary in shape When an enzyme and substrate bind they form an enzyme-substrate complex where the active site undergoes a conformational change to accommodate the substrate, and the substrate undergoes a small change in turn thee induced fit model states that an enzyme undergoes a conformational change to become complementary in shape to the substrate Activation energy defined as the minimum amount of energy required to energise atoms or molecules to a state where they can undergo a chemical transformation anabolic reaction is when two or more smaller molecules combine to form a larger one catabolic reaction is a larger molecule turning into two or more smaller molecules In a biochemical pathway, one enzyme will function to catalyse a substrate into a product, which will then become the substrate of a second enzyme proteome the complete array of proteins produced by a single cell or an organism in a particular environment An organism’s proteome consists of a large group of diverse proteins with different structures and functions  DOT POINT 7 - The role of the rough endoplasmic reticulum, Golgi apparatus and associated vesicles in the export of proteins from a cell via the protein secretory pathway Proteins produced in ribosomes at the rough endoplasmic reticulum that are intended for export must be transferred to the Golgi apparatus and then to secretory vesicles ○ vesicles membrane-bound sacs found within a cell, such as secretory vesicles, which are involved in the export of proteins EXOCYTOSIS A type of bulk transport where the contents of a vesicle are released from a cell ○ bulk transport a type of active transport that uses vesicles to move large molecules or groups of molecules into or out of the cell also a form of active transport, and therefore, the process of exocytosis requires the input of energy Steps involved ○ A vesicle containing secretory products is transported to the plasma membrane ○ The membrane of the vesicle fuses with the plasma membrane ○ The secretory products are released from the cell into the extracellular environment. PROTEIN SECRETORY PATHWAY Ribosomes The ribosomes are the sites of protein synthesis They assemble polypeptide chains from amino acids by translating mRNA Rough endoplasmic reticulum Proteins produced by ribosomes on the endoplasmic reticulum are generally exported from the cell The environment inside the rough endoplasmic reticulum allows for the correct folding of the newly formed polypeptide chain before being passed to the Golgi apparatus (folds and transports) A transport vesicle containing the protein buds off the rough endoplasmic reticulum and travels to the Golgi apparatus. The vesicle fuses with the Golgi membrane and releases the protein into its lumen Golgi apparatus packaged into secretory vesicles for export or released directly into the cytosol for use by the cell Modifies and packages proteins Vesicles Secretory vesicles containing proteins for export bud off the Golgi apparatus and travel through the cytoplasm, fusing with the plasma membrane. This releases the proteins contained from within, into the extracellular environment through the process of exocytosis mitochondria are the site of ATP synthesis and provide the energy required to move vesicles around the cell and modify the proteins produced  [CHAPTER 2] DOT POINT 8 - The use of enzymes to manipulate DNA, including polymerase to synthesise DNA, ligase to join DNA and endonucleases to cut DNA POLYMERASES polymerase an enzyme that synthesises a polymer from monomers, such as forming a DNA strand from nucleic acids In order for DNA polymerase to synthesise DNA, the double helix needs to be unzipped and made single stranded synthesises polymer chains from monomer building blocks used in the replication or amplification of DNA require a primer to attach to the start of a template strand of DNA. ○ primers are short single-stranded chains of nucleotides that are complementary to the template strand Once attached to the primer, the polymerase enzyme can read and synthesise a complementary strand to the template strand in a 5’ to 3’ direction Always add to the growing 3’ end of the growing polymer Reverse transcriptase When small sections of DNA or specific genes are required to be synthesised, using the mRNA as a template can be more useful mRNA needs to isolated from the specific cells in which the gene concerned is active the enzyme reverse transcriptase uses the mRNA as a template to build a single-stranded DNA with a complementary base sequence Polymerase then builds the second strand known as complementary DNA (cDNA) ○ complementary DNA (cDNA) a strand of DNA that has complementary bases to the opposite strand and is usually produced through reverse transcription LIGASES ligase an enzyme that joins molecules, including DNA or RNA, together by catalysing the formation of phosphodiester bonds catalyses the joining of pieces of double-stranded DNA at their sugar–phosphate backbones by strong phosphodiester (covalent) bonds can join together any blunt or sticky ends ENDONUCLEASES endonuclease an enzyme that breaks the phosphodiester bond between two nucleotides in a polynucleotide chain When they target specific recognition sites, they are known as restriction endonucleases ○ restriction endonuclease any enzyme that acts like molecular scissors to cut nucleic acid strands at specific recognition sites. Also known as a restriction enzyme  they cleave the phosphodiester bond of the sugar-phosphate backbone that holds DNA nucleotides together Generally, recognition site sequences are palindromes, which means the 5’ to 3’ sequence of the template strand is the same as the 5’ to 3’ sequence of the non-template strand Create sticky ends or blunt ends ○ sticky end the result of a staggered cut through doublestranded DNA by an endonuclease resulting in overhanging nucleotides ○ blunt end the result of a straight cut across the double-stranded DNA by an endonuclease resulting in no overhanging nucleotides DOT POINT 9 - The function of CRISPR-Cas9 in bacteria and the application of this function in editing an organism’s genome Clustered Regularly Interspaced Short Palindromic Repeats naturally occurring sequence of DNA found in bacteria that plays an important role in their defence against viral attacks from bacteriophages ○ bacteriophage a virus that infects prokaryotic organisms CRISPR are short repeated segments of DNA, with each repeated segment being separated by a length of spacer DNA. Cas9 is a specific enyzme (specifically an endonuclease) that can cut DNA associated with CRISPR is a gene editing tool ○ Inexpensive (cheap) ○ Easy to use and efficient ○ Precise gene editing When a bacterium encounters a virus, it takes a ‘mugshot’ of it by storing some of the viral genetic material within the bacterium’s own genome Next time the virus invades, the bacterium transcribes the ‘mugshot’ DNA and attaches it to an endonuclease called Cas9. The transcribed mugshot is complementary to the viral DNA, so it ensures that the Cas9 only destroys the invading virus rather than any bacterial nucleic acids. In this way, we can say that CRISPR-Cas9 functions as a primitive adaptive immune system in bacteria, defending them from viral invasion Cas-9 is an endonuclease (restriction enzyme) with 2 active sites ○ stores viral DNA found in ‘spacer’ regions between the clustered regularly interspaced short palindromic repeats (CRISPR) PAM (Protospacer adjacent motif) (nGG)- 2-6 nucleotides found immediately next to the DNA targeted by Cas9 which allow Cas9 to bind to DNA plays an essential role in distinguishing self from non-self When Cas1 or Cas2 come across the PAM, they are signalled to extract a protospacer from invading DNA. The enzymes cut the viral DNA just before the PAM, so the PAM does not get included in the final protospacer  Bacteria never have a PAM sequence in the CRISPR repeats of their own DNA, ensuring hat Cas9 cannot cut up a bacterium’s own DNA Steps Exposure – the bacteriophage injects its DNA into a bacterium, which identifies the viral DNA as a foreign substance. ○ Cas enzymes cut out a short section of the viral DNA (typically ~30 nucleotides long), known as a protospacer. ○ This protospacer can then be introduced into the bacterium’s CRISPR gene and become a spacer Expression – the CRISPR spacers are transcribed along with half a palindrome from the repeat either side of it, and converted into an RNA molecule known as guide RNA (gRNA). ○ gRNA binds to Cas9 to create a CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that is complementary to the gRNA. ○ gRNA forms a hairpin loop-like structure from the transcribed palindromic repeats either side of the spacer. guide RNA (gRNA) RNA which has a specific sequence determined by CRISPR to guide Cas9 to a specific site Extermination - The CRISPR-Cas9 complex then scans the cell for invading bacteriophage DNA that is complementary to the ‘mugshot’ on the gRNA. ○ When it does, Cas9 cleaves the phosphate-sugar backbone to inactivate the virus. ○ Cas9 contains two active sites to cut both strands of DNA and create blunt ends. GENE EDITING genome editing a process by which changes are made to the nucleic acid sequence of genes; also termed gene editing CRISPR-Cas9 can induce genetic changes by cutting DNA at a location specifically chosen by scientists, who make a synthetic sgRNA, known as single guide RNA (sgRNA), to guide Cas9 ○ single guide RNA (sgRNA) guide RNA utilised by scientists to instruct Cas9 to cut a specific site when using CRISPR-Cas9 in gene editing  This cutting of the DNA creates an opportunity for nucleotides to be added, removed, or substituted into the selected sequence. In turn, this can knockout, enhance, or change the function of a gene ○ gene knockout a technique in gene editing where scientists prevent the expression of a target gene to understand its function in an organism Steps 1. Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA that scientists wish to cut. 2. A Cas9 enzyme is obtained with an appropriate target PAM sequence. 3. Cas9 and sgRNA are added together in a mixture and bind together to create the CRISPR-Cas9 complex. 4. The sgRNA-Cas9 mixture is then injected into a specific cell, such as a zygote. 5. The Cas9 finds the target PAM sequence and checks whether the sgRNA aligns with the DNA. 6. Cas9 cuts the selected sequence of DNA. 7. The DNA has a blunt end cut that the cell will attempt to repair. 8. When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site. Scientists may inject particular nucleotide sequences into the cell with the hope that it will ligate into the gap. LIMITATIONS To induce substitution mutations or knock-in a new segment of DNA, scientists must introduce the nucleotide sequence they wish to add into the cell and hope it is taken up by the DNA repair machinery. This can be difficult to achieve with precision and is not consistently successful  To successfully alter an organism’s genome using CRISPR-Cas9 technologies, scientists must treat an embryo prior to the cells differentiating, as this will ensure every cell in the organism is altered. Some groups are concerned that scientific research on embryos does not respect the sanctity of human life it is currently illegal to implant genetically modified embryos into human females, and to allow the embryo to develop and be born Safety – the possibility of off-target cleavages (edits in the wrong place) and mosaics (some cells containing edited genomes, others not) mean that many scientists are hesitant to use CRISPR outside of research. Informed consent – scientists cannot get consent from embryos to edit their genes. If the embryo goes on to be born and one day has children of its own, these children also will never have consented to scientists interfering with their genome. Inequality – there is concern that only wealthy people will be able to afford to use CRISPR to treat genetic conditions or otherwise change their genes. Discrimination – CRISPR may be a threat to those who are judged by society as biologically inferior, when in fact those individuals do not feel they need ‘fixing’ at all. DOT POINT 10 - Amplification of DNA using polymerase chain reaction and the use of gel electrophoresis in sorting DNA fragments, including the interpretation of gel runs for DNA profiling POLYMERASE CHAIN REACTION polymerase chain reaction (PCR) a laboratory technique used to produce many identical copies of DNA from a small initial sample ○ a DNA manipulation technique that amplifies DNA by making multiple identical copies Taq polymerase an enzyme used in PCR that adds free nucleotides to the single stranded DNA in order to synthesise a new strand Purpose- The polymerase chain reaction amplifies a sample of DNA to increase the quantity of DNA available After each cycle of the polymerase chain reaction, the amount of DNA present is doubled ○ 2^n (where n is the number of cycles) Ingredients Taq polymerase - an enzyme used in PCR that adds free nucleotides to the single stranded DNA in order to synthesise a new strand Buffer - provide a suitable chemical environment for the activity of Taq polymerase by maintaining pH and providing DNA sample - provide a template to produce copies of in PCR (gets denatued and amplified) Primers - bind to the single-stranded DNA and to provide a point in which DNA synthesis can be initiated and designate the sequence to be copied. Free nucleotides - To be added by Taq polymerase to produce the new DNA strand PCR tube - To provide a vessel for the PCR reaction to occur. The tube will contain the DNA sample, polymerase, primers, nucleotides and buffer. Steps Denaturing - DNA is heated to approximately 90–95 °C to break the hydrogen bonds between the complementary bases and separate the strands, forming single-stranded DNA (occurs for 1 minute) Annealing - the single-stranded DNA is cooled to approximately 50–55 °C to allow the primers to bind to complementary sequences at the 3’ end on the single-stranded DNA (occurs for 2 minutes)  ○ forward primer a DNA primer that binds to the 3’ end of the template strand and reads the DNA in the same direction as RNA polymerase ○ reverse primer a DNA primer that binds to the 3’ end of the coding strand and reads the DNA in the reverse direction to RNA polymerase ○ having these two primers is necessary as the 5’ ends of both the template and coding strands are different. As Taq polymerase only functions towards the 3’ end, a primer is needed for both strands to facilitate this directionality Elongation - DNA is heated again to 72 °C, which allows Taq polymerase to work optimally. Taq polymerase binds to the primer, which acts as a starting point, and uses the free nucleotides to synthesise new complementary strands to each of the single template strands (occurs for 1 minute) Process is repeated, doubling the amount of DNA every time, where everything thing, including mutations, will be copied GEL ELECTROPHORESIS gel electrophoresis a technique that separates DNA fragments based on their molecular size through an electric field DNA fingerprinting technique for identifying DNA from different individuals based on variable numbers of tandem repeats of short DNA segments near the ends of chromosomes DNA sequencing identification of the order or sequence of bases along a DNA strand typically used after a sample of DNA has been cut up using restriction endonucleases or after a short sequence of DNA has been amplified using the polymerase chain reaction Steps 1. The DNA smaple with fragments of varying sizes in combined with DNA loading dye and placed in the wells at one end of the agarose gel, which is immersed in a buffer solution 2. The gel is then exposed to an electric field with positive pole (anode) at the far end and the negetaive pole (cathode) at the starting origin 3. Smaller fragments move through the agarose gel faster compared to larger fragment and eventually settle into bands 4. The gel can be interpreted after being observed under a UV light  standard ladder a mixture of DNA fragments of known length that are used to infer the size of fragments in a sample agarose gel a sponge-like gel used in gel electrophoresis that contains pores for DNA fragments to move through buffer an ion-rich solution that carries electrical current through the agarose gel buffer solution solution that helps maintain pH (agarose gel is submerged in buffer) long fragments of DNA collect in bands of DNA near the well, while shorter fragments form bands further from the well A standard ladder of DNA fragments of known sizes is usually run through the gel at the same time as the unknown DNA samples as it helps compare the positions of their bands with those of the known standard The distance the band travels is also influenced by the concentration or viscosity of the agarose and the specific voltage or power used Once the gel run is complete, the separated DNA bands must be made visible either through the use of a dye or a labelled probe ○ probe single-stranded segment of DNA (or RNA) carrying a radioactive or fluorescent label with a base sequence complementary to that in a target strand of DNA Factors affecting movement voltage – the stronger the electric force generated by the electrodes the further DNA travels towards the positive electrode gel composition – gels with a greater density and agarose concentration increase the difficulty for larger fragments to move through buffer concentration – the greater the concentration of ions in the buffer the more the electric current is conducted through the gel, which causes DNA to move further down the lane time – the longer the electric current is applied, the further the DNA will travel. Note: if too much time passes, the DNA may move out of the gel. APPLICATIONS genetic testing screening an individual’s DNA for anomalies that may make them susceptible to a particular disease or disorder DNA profiling the process of identification on the basis of an individual’s genetic information can amplify DNA samples using the polymerase chain reaction until we have a larger total amount of testable genetic material short tandem repeats (STR) short, repeated sequences of nucleotides found in the noncoding regions of nuclear DNA STRs are small sections of repeated nucleotides that vary in length between people and are found in the non-coding areas of autosomal chromosomes not affected by natural selection Useful in parental testing as the child must inherit half of their STRs from each one of their parents If the individual is heterozygous for an STR their gel will have two bands, whereas if they are homozygous their gel will only have one thick band  DOT POINT 11 - The use of recombinant plasmids as vectors to transform bacterial cells as demonstrated by the production of human insulin plasmid a small circular piece of double-stranded DNA that is able to reproduce independently and may be taken up by cells (usually bacteria) in addition to chromosomal DNA Once a plasmid is edited to integrate a target gene, it is referred to as a recombinant plasmid ○ recombinant plasmid a circular DNA vector that is ligated to incorporate a gene of interest Bacteria then take up these recombinant plasmids from the environment in a process called bacterial transformation MAKING RECOMBINANT PLASMIDS Steps 1. The DNA of the plasmid is cut using a specific cutting enzyme (endonuclease) in order to create sticky ends. This changes the plasmid from circular to linear. 2. The foreign DNA fragments (gene of interest) are prepared using the same endonuclease so that is has sticky ends complementary to the cut plasmid. Often, the process of reverse transcription is used to create these foreign fragments to ensure that non-coding introns are not included. 3. The gene of interest and the plasmids are mixed, and, in some cases, their ‘sticky ends’ pair by using weak hydrogen bonds. A recombinant plasmid has been created. (Other pairings will also occur, such as cut plasmids resealing themselves so that they are not recombinant plasmids). 4. The joining enzyme, ligase, is added and this makes the joins permanent through covalent bonding. Gene of interest a gene scientists want to be expressed in recombinant bacteria. This gene often encodes a protein we wish to produce in commercial quantities ○ Must not contain introns ○ DNA is amplified before being inserted intoa vector vectors an agent or vehicle used to transfer pathogens or genes between cells and organisms plasmid vector a piece of circular DNA that is modified to be an ideal vector for bacterial transformation experiments ○ A plasmid vector is selected into which the gene of interest will be inserted ○ origin of replication (ORI) a sequence found in prokaryotes that signals the start site of DNA replication ○ antibiotic resistance genes a gene governing antibiotic resistance, with the particular form of the gene being the allele for resistance to an antibiotic ○ reporter gene gene with an easily identifiable phenotype that can be used to identify whether a plasmid has taken up the gene of interest  role is to distinguish between a recombinant and non-recombinant plasmid A screening marker allows for the confirmation that a plasmid is actually recombinant UPTAKE RECOMBINANT PLASMIDS Bacterium selected should be appropriate: ○ Needs to be able to reproduce quickly ○ Needs to be harmless to individuals working with it ○ E.coli is commonly used Electropolation a technique that uses brief exposure of host cells to an electric field to enable the entry of segments of foreign DNA into the cells ○ Cells are briefly placed in an electric field that shocks them and appears to create holes in their plasma membranes, allowing the plasmid entry heat shock a method that involves rapidly increasing and decreasing the temperature to increase membrane permeability in order to enhance the likelihood of bacterial transformation ○ The bacterial cells are suspended in an ice-cold salt solution and then transferred to 42 °C for less than one minute. This treatment appears to increase the fluidity of the plasma membranes of the bacterial cells and increases the chance of uptake of plasmids Steps of heat shock 1. The bacterial culture is placed in an ice bath and chilled 2. Recombinant plasmids with the tetracycline resistance allele TetR are added to the bacterial culture and chilled. 3. The bacteria and plasmid mix are placed in hot water at 42 °C for 50 seconds, producing a heat shock. This is the stage when the plasma membranes of the bacterial cells are altered, increasing the chance of uptake of plasmids by the cells. 4. The mix is returned to an ice bath for two minutes 5. The bacteria are plated on an agar plate containing the antibiotic tetracycline and incubated at 37 °C overnight. Bacteria that have not taken up the plasmids are killed by the tetracycline. Bacterial cells that have taken up the plasmids will be selected as they will survive and replicate The importance of recombinant plasmids is that the foreign DNA they incorporate can come from any source: it may be a human gene, a plant gene, a jellyfish gene, a yeast gene and so on. The importance of transformed bacteria is that they express the foreign gene in their phenotype. Checking that the plasmid has the gene One common technique to ensure that the gene of interest that has been inserted is using a screening marker such as lacZ ○ LacZ codes for an enzyme known as β-galactosidase (beta-galactosidase), which converts a colourless substrate (X-gal) to a blue product. If the gene of interest is incorporated into the plasmid at the BamHI recognition site, the enzyme that makes the blue product is lost because the DNA of this segment is disrupted If the gene of interest is not present, the enzyme is produced and the blue colour can be produced bacteria are grown on an agar plate containing X-gal, those containing the gene of interest appear white and can be used for further replication Those that are blue do not contain the gene of interest, so are discarded Checking that the bacteria contains the plasmid under a specific antibiotic, bacteria without a plasmid conferring resistance will not survive meaning that the bacteria has not taken up the plasmid bacteria that has taken up the recombinant plasmid will survive with the antibiotic as the plasmid contains the antibiotic resistance gene INSULIN insulin a hormone that allows for glucose to enter cells, reducing blood glucose levels Insulin is an important hormone that is responsible for regulating our blood glucose levels People with diabetes do not naturally produce or respond to insulin and require it to be administered artificially into their body insulin protein has a quaternary structure consisting of two polypeptide chains known as the alpha and beta subunits (not to be confused with alpha helices and beta-pleated sheets). to produce insulin, we require two different recombinant plasmids and thus two different transformed bacteria samples - one producing the alpha subunit and one producing the beta subunit Steps Creating the recombinant plasmid 1. Plasmid vectors were prepared which contained the ampR gene to encode for antibiotic resistance to ampicillin and tetR to encode for antibiotic resistance to tetracycline. tetR acted as a reporter gene and had a specific recognition site to one of the restriction endonucleases used in step 2 inside it. 2. Two plasmid vectors were used - one for insulin subunit A and one for insulin subunit B. Using the two restriction endonucleases EcoRI and BamHI, both plasmid samples, the insulin A subunit gene, and the insulin B subunit gene, were all cut to form complementary sticky ends. DNA ligase was then used to reestablish the sugar-phosphate backbone and create two different recombinant plasmids. Creating transformed bacteria 3. The plasmids were added to a solution of E. coli bacteria and some of the recombinant plasmids were taken up by the bacteria. To determine which bacteria successfully took up plasmids, the bacteria cultures were spread and incubated onto agar plates containing the antibiotic ampicillin. Colonies that formed were identified to have taken up a plasmid 4. Tto determine which colonies contained bacteria that took up recombinant plasmids some of each of the colonies from the ampicillin plate were spread onto agar plates containing tetracycline. If the bacteria were not resistant to tetracycline, then it was known that they contained recombinant plasmids. This is because  the insertion of the insulin subunit gene interrupted the tetR gene, thus making bacteria with recombinant plasmids susceptible to tetracycline. These plasmids were then collected. 5. The plasmids were then cut open once again using EcoRI, to insert another gene called lacZ (minus its stop codon). lacZ produces ß-galactosidase, a large enzyme. 6. The recombinant plasmids containing lacZ were added to a new solution of E. coli bacteria and some of these new recombinant plasmids were taken up by the bacteria. 7. To determine which bacteria successfully took up the new recombinant plasmids, a test was performed that relied on the function of ß-galactosidase. The E. coli were plated on agar plates containing ampicillin and X-gal. Colonies that grew and were blue in colour were identified as containing recombinant plasmids due to the presence of ß-galactosidase. These bacteria were capable of producing the insulin subunit proteins that were attached to ß-galactosidase. Protein production and extraction 8. Transformed bacteria that contained the recombinant plasmid were then placed into conditions to exponentially reproduce before their membranes were broken down, and the insulin subunit and ß-galactosidase fusion proteins were extracted. The compound cyanogen bromide was added to break down the methionine that was added at the start of the insulin gene. In doing so, it separated the insulin subunit from the ß-galactosidase, allowing for the isolation and purification of the insulin subunit. a. fusion protein a protein made when separate genes have been joined and are transcribed and translated together 9. The two insulin chains were then mixed together, which allowed the connecting disulphide bonds to form and create functional human insulin. DOT POINT 12 - The use of genetically modified and transgenic organisms in agriculture to increase crop productivity and to provide resistance to disease GENETICALLY MODIFIED ORGANISMS TRANSGENIC ORGANISMS Genetically modified organisms (GMOs) organisms Transgenic organisms a genetically modified organism whose genomes are altered through the use of genetic that contains foreign genetic material from a separate engineering technology species organism that receives the altered gene/s is Comprise a subgroup of GMOs that includes those referred to as the host organism GMOs in which the alteration to the genome involves the genetic material from a different GMOs have combination of genes that do not species. occur in natural populations but are created able to produce proteins that were not previously through the means of genetic engineering part of their species’ proteome due to their genome The genetic alteration achieved through genetic being altered with foreign DNA engineering may involve genes: results in an organism that contains foreign DNA ○ being silenced transplanted from a separate species ○ inserted into the genome ○ removed from the genome ○ altered by replacing nucleotides. So, it follows that all transgenic organisms are For an organism to be GMO 🡪 any gene or DNA segment GMOs, but not all GMOs are transgenic. that is added through genetic engineering should be heritable; that is, it should be able to be passed on to the next generation.  Genetic engineering refers to the alteration of an organism’s genome using genetic recombination technologies ○ genetic engineering the process of using biotechnology to alter the genome of an organism, Genetic typically with the goal of conferring some desirable trait APPLICATIONS IN AGRICULTURE Increasing crop productivity Modifications include improved photosynthetic efficiency, greater crop yields and faster growth rates includes the use of CRISPR-Cas9 technologies to modify plant genomes Increasing disease resistance developing crops that are less impacted by harmful plant pathogens, scientists can improve global food security by minimising crop destruction and the spreading of disease resistance to other damaging environmental factors such as drought and herbivorous pests can also be increased Genetically engineering plants that are resistant to disease is a path to reducing the risk of crop loss and ensuring a stable supply of food for the world ISSUES Implication Pros Cons Biological GM crops have better crop productivity GM crops may lose their effectiveness if than non-GM crops, means that more weeds or pests evolve resistance. food can be grown using less land, Widespread use of GM crops could result in reducing habitat loss the loss of genetic diversity within crop Insect-resistant GM plants require fewer populations. pesticides, better for the environment Cross-pollination between GM crops and GM foods can be made to have improved wild species or weeds may cause genes to nutritional content, improving the health spread and lead to unforeseen of individuals consequences. Social Increased crop productivity means more Having to buy new seeds each season may food can be produced, leading to better be costly for farmers. food security. Complex legal issues surrounding the use of Crops that are able to grow in more GM products may cause farmers undue adverse conditions (e.g. drought-tolerant stress and anxiety related to regulation. corn) protect against famine, improving There are strict packaging and marketing food security. regulations for GMO producers that may not Increased crop yields result in larger be complied with if either the producer or profits for farmers. consumer are undereducated on these regulations. Ethical Some people believe that using genetic Some people consider GMOs to be modification is an ethical imperative unnatural, or like we are ‘playing God’. given the potential for widespread Some people believe that GM foods are benefits, including nutrition, wealth, and unsafe to eat and choose not to eat them as a the overall health of humanity, especially result. This is especially true if there is lack in developing nation of long-term evidence of healthy use. Some people believe that genetically modifying animals for human benefit is inhumane The fact that companies can own the rights to GM crops is considered by some to be unethical due to companies possibly making unfair demands of farmers. This ownership power divide can materialise in a range of ways, including the following: – ○ Cross-pollination of non-GM crops by nearby GM crops could result in the non-GM farmer being sued by the patent-owner. – ○ Farmers can’t reuse seeds from some GM crops and must buy new expensive seed supplies each year from biotechnology companies AOS 2 – HOW ARE BIOCHEMICAL PATHWAYS REGULATED? [CHAPTER 3] DOT POINT 1 - the general structure of the biochemical pathways in photosynthesis and cellular respiration from initial reactant to final product biochemical pathway is a series of linked biochemical reactions that start with an initial reactant that is converted in a stepwise fashion to a final product ○ Each step in a biochemical pathway requires the activity of a specific enzyme metabolism the total activity of the reactions of all biochemical pathways in a living organism enzyme a protein that acts as a biological catalyst, speeding up reactions without being used up reactants a substance that is changed during a chemical reaction ○ Chemically changed to form products reactant molecules in each step can also be called the substrates for their particular enzymes ○ substrates a compound on which an enzyme acts products the compound that is produced in a reaction endergonic a chemical reaction that is energy-requiring exergonic a chemical reaction that is energy-releasing  Anabolic (building up) Catabolic (breaking down) assemble simple molecules into more complex Pathways break down complex molecules into molecules. more simple molecules building complex molecules from simple ones Energy-releasing or exergonic. requires an input of energy Eg. Aerobic cellular respiration - glucose Energy-requiring or endergonic. molecules are broken down into carbon Eg. Photosynthesis - glucose molecules are dioxide and water synthesised from carbon dioxide and water produce a net release of energy as the energy using radiant energy from the Sun level of the initial reactants is higher than that require energy, so the energy level of the of the final products initial reactants is lower than that of the final products DOT POINT 2 - The general role of enzymes and coenzymes in facilitating steps in photosynthesis and cellular respiration Enzymes speed up the rates at which the products of reactions are formed by lowering the activation energy needed for reactions In the presence of an enzyme, the activation energy needed to start a reaction is much lower than that for an uncatalysed reaction ○ Since less energy is required for an enzyme-catalysed reaction to get started, more substrate molecules and enzymes will have sufficient energy to react when they collide Enzymes consist of one or more polypeptide chains When a substarte joins to an active site of an enzyme it temporarily forms an enzyme-substrate complex ○ enzyme–substrate complex transient compound produced by the bonding of an enzyme with its specific substrate shape of the active site of an enzyme is not rigid and it can adjust to the shape of its substrate ENZYMES IN PHOTOSYNTHESIS/ CELLULAR RESPIRATION Photosynthesis- anabolic pathway Having enzymes regulate each step in photosynthesis ensures reactions are sped up and controlled, so plants can metabolise efficiently cellular respiration- catabolic pathway  coenzymes an organic molecule that acts with an enzyme to alter the rate of a reaction ○ Subset of cofactors Cofactor a non-protein molecule or ion that is essential for the normal functioning of some enzymes Inorganic cofactors do not contain carbon and include metal ions Organic cofactors are small non-protein organic molecules that are essential for the function of particular enzymes During the reaction, the coenzyme binds to the active site, donates energy or molecules, and then cannot be immediately reused After the reaction, the coenzyme leaves the enzyme and is recycled by accepting more energy, so it can then go on to assist in more reactions referred to as the cycling of coenzymes and is integral to certain biochemical processes transfer atoms or groups of atoms, such as hydrogens, phosphate groups and acetyl groups energy transfers Coenzymes exist in two inter-convertible forms: ○ a high energy form that is loaded with a group that can be transferred ○ a lower energy form that is unloaded loaded the form of coenzymes that can act as electron donors unloaded a form of coenzymes that can act as electron acceptors COENZYMES IN CELLULAR RESPIRATION non-protein organic molecules Coenzyme Description NAD NAD+ the unloaded form of NADH, which can accept hydrogen ions and electrons during cellular respiration When a substrate needs to give up electrons and hydrogen ions (or is 'oxidised'), NAD+ is able to accept these, becoming NADH As NAD transitions between its loaded and unloaded forms, it switches from being a helper for the group of enzymes that catalyse reactions where electrons are lost (‘reduction’) to being a helper for those enzymes catalysing reactions where electrons are gained (‘oxidation’). NADH the loaded form of NAD+ which can donate hydrogen ions and electrons during cellular respiration FAD FAD a coenzyme with a similar function to NAD, accepting hydrogen ions and electrons  during cellular respiration (unloaded) FAD plays a role in the second stage of cellular respiration (the Krebs cycle) and accepts high-energy electrons FADH2 the loaded form of FAD, which can donate hydrogen ions and electrons during cellular respiration FADH2 plays a role in the last stage of the cellular respiration pathway (the electron transport chain) as a donor of these high-energy electrons into a chain of electron acceptors ATP ATP adenosine triphosphate; the common source of chemical energy for cells ○ energy rich and it is a major player in transferring energy within cells ○ This energy is made available when the last phosphate group of ATP is removed, creating ADP ADP adenosine diphosphate; a coenzyme that accepts a phosphate group to form ATP ADP is a coenzyme helper to some enzymes when it acts as an acceptor of a phosphate group from the substrate of an enzyme to form ATP ADP + Pi ⇋ ATP Coenzyme A Coenzyme A a coenzyme that aids pyruvate (CoA) decarboxylase during cellular respiration, accepting an acetyl group role is to accept the acetyl group and transfer it to another acceptor molecule. ○ important in the second stage of cellular respiration, the Krebs cycle COENZYMES IN PHOTOSYNTHESIS Coenzyme Description NADPH NADPH the loaded form of NADP+, which can donate hydrogen ions and electrons during photosynthesis NAPDH is formed from NADP+ using high-energy electrons initially produced by uptake of the radiant energy of sunlight NADP+ the unloaded form of NADPH, which can accept hydrogen ions and electrons during photosynthesis NADPH is used in the second stage of the photosynthesis pathway when they donate electrons and transfer energy required by enzymes in this pathway as sugars are built. In doing so, NADPH reverts to NADP+. ATP high-energy ATP is formed from ADP and the energy required is initially derived from the radiant energy of sunlight. ATP can then release its energy to help enzymes involved in energy-requiring reactions that build sugar molecules from carbon dioxide DOT POINT 3 - The general factors that impact on enzyme function in relation to photosynthesis and cellular respiration: changes in temperature, pH, concentration, competitive and non-competitive enzyme inhibitors TEMPERATURE  optimum temperature the temperature at which the rate of reaction catalysed by an enzyme is at its highest It can be seen that when temperature first increases, the reaction rate also increases. This is because the reactants absorb heat energy and move faster, allowing them to collide and overcome the activation energy The enzymes found within the human body have an optimal temperature range of 36–38 °C, Decrease ○ As the temperature drops below the optimum, the rate of reaction reduces progressively and molecular movements slow, resulting in fewer collisions between substrates and enzyme, and fewer molecules have sufficient energy to interact ○ enzymes can regain functionality when reheated as significant denaturation does not occur at low temperatures ○ as the temperature decreases towards the lower limit of the tolerance range, enzyme activity slows until freezing occurs, causing loss of function. This freezing is reversible as it does not cause an irreversible conformational change Increase ○ As the temperature increases above the optimum, the rate of reaction dips quickly due to the denaturation of the enzymes which causes a conformational change in its active site causing the substrate to no longer fit ○ Change is irreversible PH Different enzymes have corresponding optimal pH denaturation of an enzyme occurs if it is exposed to an environment that is either above or below the optimal pH. That is, both overly acidic or basic environments can denature a given enzyme As the pH moves progressively from the optimum value — either up or down — various ionic bonds that contribute to the shape of the enzyme can be altered, and the bonds that temporarily hold a substrate in place in the active site can be altered. If this happens, the enzyme is less able to combine with its substrate, and its activity is reduced or shut down SUBSTRATE CONCENTRATION For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration — but only up till the saturation point. Beyond this, any further increase in substrate concentration produces no significant change in reaction rate, as all the active sites of the enzyme molecules at any given moment are occupied by substrate molecules Eventually, the maximum reaction rate is reached and, at any time after that, the active sites of all enzyme molecules are saturated by substrate molecules saturation point the point at which a substance (e.g. an enzyme) cannot receive more of another substance (e.g. a substrate)  Before the graph plateaus, we can say that the substrate concentration is a limiting factor in the reaction, or, more specifically a limiting reagent limiting factor a factor that prevents the rate of reaction from increasing limiting reagent a reactant that prevents the rate of reaction from increasing ENZYME CONCENTRATION If the enzyme concentration is high, then the reaction rate will be high. This is due to the large number of active sites available for the substrate to bind to If the enzyme concentration rises (while the substrate concentration is kept constant), then the reaction rate will increase. This is true until enzymes are in excess, at which point the reaction rate will plateau regardless of any continued increase in enzyme concentration (point of saturation is reached where more enzyme will not make the reaction rate occur any faster) COMPETITVE INHIBITION competitive inhibitor inhibition in which a molecule binds to the active site of an enzyme instead of the usual substrate shape must be complementary to the enzyme’s active site and therfore must have a similar shape to the substrate prevent the usually substrate from binding Adding more substrate molecules increases the chance that a random collision with the active site will be with a substrate molecule rather than a molecule of the competitive inhibiton  NON-COMPETITVE INHIBITION non-competitive inhibition inhibition in which a molecule binds to the allosteric site of an enzyme causing a conformation change in the active site allosteric site a region on an enzyme that is not the active site where a molecule can bind and alter shape of the enzyme conformational change in the active site’s structure prevents the substrate from binding to it, preventing the reaction from occurring Increasing substrate concentration will not remove the effect of the non-competitive inhibition. As a result, a fixed percentage of the enzyme molecules are always inactivated by a non-competitive inhibitor REVERSIBLE AND IRREVERSIBLE INHIBITION Reversible reversible inhibition enzyme inhibition that involves weaker bonds that can be overcome reversible inhibitors typically slow the rate of a given enzyme-catalysed reaction, but do not stop it indefinitely inhibitor can have its effects overcome by increasing the amount of substrate present Irreversible Both competitive and non-competitive inhibition of enzymes are processes that can be reversed form strong bonds that are unbreakable enzyme is unable to bind with any substrate or catalyse any reactions indefinitely regardless of how much extra substrate is present, the reaction can never occur BIOCHEMICAL PATHWAYS biochemical pathways are precisely coordinated and regulated so that a balance is established between energy production and energy needs of cells Controlling enzyme activity is a major means by which regulation of biochemical pathways is achieved Pathways may be down-regulated by slowing or stopping the activity of specific enzymes in the pathway. Pathways may be up-regulated by increasing the activity of specific enzymes. Allosteric regulation allosteric regulation the control of the reaction rate of enzymes through conformational changes in enzymes Allosteric regulation of one or more enzymes in the pathway is controlled through conformational changes in enzymes Not permanent change and can be reversed allosteric inhibitors molecules that bind to the allosteric site of an enzyme and stop enzyme activity ○ binding produces a change of shape in the enzyme that stops enzyme activity; they act like an OFF switch allosteric activators molecules that bind to the allosteric site of an enzyme and increase enzyme activity  ○ shape change resulting from the binding produces an increase in enzyme activity; they act like an ON switch Feedback Inhibition feedback inhibition inhibition occurs when the end product of a pathway inhibits an enzymes earlier in the pathway as a negative feedback mechanism; also known as end-product inhibition end product of a metabolic pathway acts as an inhibitor of the key enzyme that catalyses the first step in a pathway [CHAPTER 4] DOT POINT 4 - Inputs, outputs and locations of the light-dependent and light-independent stages of photosynthesis in C3 plants (details of biochemical pathway mechanisms are not required) Photosynthesis process by which plants use the radiant energy of sunlight trapped by chlorophyll to build carbohydrates from carbon dioxide and water Plant cells harness light energy to produce glucose via the following steps: 1. Sunlight excites an electron within chlorophyll in the grana, causing water from the roots to split into oxygen and hydrogen. 2. The excited electron and the hydrogen ion from the split water facilitate the production of ATP and NADPH. These molecules are essential for the Calvin cycle. 3. The oxygen from the split water is released out of the chloroplast as a by-product. 4. Carbon dioxide enters via the stomata. With the help of ATP and NADPH, the carbon from carbon dioxide undergoes a series of reactions in the Calvin cycle. 5. Eventually, a molecule is produced that contributes to the formation of glucose and the cyclic reaction continues. Some water is also formed in this stage. The essential purpose of photosynthesis is to capture sunlight and transform this energy into the concentrated chemical energy of organic sugar molecules — that is, to make sugars, such as glucose, from sunlight CHLOROPLAST chloroplast a membrane-bound organelle only found in plant and photoautotroph cells that is the site of photosynthesis directly responsible for initiating photosynthesis by capturing and being energised by light energy chlorophyll a chemical found in the thylakoids of chloroplasts. It is responsible for absorbing light energy in photosynthesis thylakoids flattened membranous sacs in chloroplasts that contain chlorophyll grana stacks of flattened thylakoids; singular: granum stroma in chloroplasts, the semi-fluid substance which contains enzymes for some of the reactions of photosynthesis The light-trapping pigment, chlorophyll, is embedded in the thylakoid membranes.  The thylakoids provide a large surface area for the capture of sunlight LIGHT DEPENDENT STAGE light-dependent stage the first stage of photosynthesis where light energy is trapped by chlorophyll the light-dependent stage can only proceed in the presence of light The function is to transform sunlight energy that is captured by chlorophyll into the chemical energy of loaded coenzymes. Light energy is trapped by chlorophyll and used to split water molecules into H+ ions and oxygen The oxygen is given off in waste, released from the leaves into the air The H+ ions from water molecules are used to generate the high energy coenzyme NADPH The movement of H+ down its concentration gradient generates the high energy coenzyme ATP Location: Grana/Thylakoid membranes Inputs Outputs Sunlight Water Oxygen NADP+ NADPH ADP ATP LIGHT INDEPENDENT STAGE/CALVIN CYCLE light-independent stage the second stage of photosynthesis where carbon dioxide is used to form glucose in the stroma of a chloroplas Location: Stroma main enzyme in C3 plants is known as Rubisco, which is vital in carbon fixation ○ carbon fixation process by which atmospheric carbon dioxide is incorporated into organic molecules such as sugars Inorganic CO2 is converted into the carbon in organic molecules, a process termed carbon fixation. Carbon dioxide molecules are accepted into the Calvin cycle by organic 5C acceptor molecules. Loaded NADPH coenzymes donate hydrogens and electrons as molecules are reduced to higher energy levels. ATP supplies energy for the anabolic steps of this cycle. Glucose is formed as an output Inputs Outputs NADPH NADP+ ATP ADP CO2 Glucose Water  DOT POINT 5 - The role of Rubisco in photosynthesis, including adaptations of C3, C4 and CAM plants to maximise the efficiency of photosynthesis RUBISCO Rubisco a pivotal enzyme involved in initial carbon fixation during the light-independent stage of photosynthesis ○ ribulose bisphosphate carboxylase-oxygenase ○ Found in the stroma Carbon fixation – which refers to the conversion of CO2 and RuBP into 3-PGA. Here, we say that the carbon from the inorganic CO2 is ‘fixed’ into an organic compound carbon fixation the process in living organisms where inorganic carbon, typically within carbon dioxide, is converted into organic compounds such as glucose Sometimes rubisco binds with oxygen instead of carbon where binding with oxygen results in photorespiration ○ Ends up producing carbon dioxide instead of glucose photorespiration a process in which plants take up oxygen rather then carbon dioxide in the light, resulting in photosynthesis being less efficient Less photosynthesis means less glucose is produced, which, combined with wasted energy used in the photorespiration pathway, negatively impacts a plant’s ability to grow, survive, and reproduce Rubisco works most efficiently when: ○ carbon dioxide levels in leaves are high ○ oxygen levels are low (as happens when water is freely available) ○ when temperatures are moderate rubiscos affinity to bind to CO2 is much higher than that for O2 stomata of the plant leaves open to allow CO2 to enter the plant, while O2 and water vapour simultaneously diffuse out of the plant. ○ however, when a plant needs to conserve water it will close its stomata, causing the O2 produced during the light-dependent stage of photosynthesis to build up inside its cells, leading to increased photorespiration stoma (pl. stomata) a small pore on the leaf’s surface that opens and closes to regulate gas exchange affinity the tendency of a molecule/atom to bind or react with another molecule/atom C3 plants C3 plants plants that carry out the original Calvin cycle using Rubisco and are prone to photorespiration CO2 competes with oxygen to bind to Rubisco CO2 is converted into a C3 product by rubisco straight from the atmosphere Everything occcurs in the mesophyll cell Climate – Best adapted to cool, wet environments. E.g., plants, wheat, rye, rice cotton C3 plants use the least amount of ATP as C4 and CAM plants use an additional enzyme which requires ATP Photorespiration can arise in C3 plants in two situations: as temperatures increase ○ ability of the Rubisco enzyme to distinguish between carbon dioxide and oxygen decreases and, as a result, Rubisco will have a higher affinity to bind with oxygen  ○ Rate of photorespiration increases faster than the rate of photosynthesis. as conditions dry out ○ When conditions become dry and water availability declines, C3 plants close their stomata to prevent water loss ○ Blocks the entry of carbon dioxide (needed as input to the Calvin cycle) and limits the exit of oxygen ○ Rubisco enzyme will increasingly bind oxygen rather than bind carbon dioxide in the process of glucose production via the Calvin cycle C4 plants C4 plants plants that carry out an adapted Calvin cycle, in which carbon fixation and glucose production occur in different cells Glucose production in C4 plants is split into two stages, with carbon fixation taking place in mesophyll cells and glucose production via the Calvin cycle in bundle sheath cell Climate – Best adapted to hot, sunny, environments. E.g., maize, sugar cane Minimising photorespiration The first stage of this pathway — carbon dioxide to malic acid — takes place in leaf mesophyll cells The PEP carboxylase enzyme can only bind to carbon dioxide at its active site. Unlike Rubisco, it is not capable of binding to oxygen End product is malic acid Second stage is where the calvin cycle occurs in the bundle sheath cells where malic acid is continuously converted to pyruvate and carbon dioxide The released carbon dioxide creates a high-concentration CO2 environment in the bundle cells As in the usual Calvin cycle, the Rubisco enzyme joins carbon dioxide to an organic acceptor molecule (RuBP) that enters the Calvin cycle for glucose production. The steady production of carbon dioxide into the bundle sheath cells means that the Rubisco enzyme will preferentially bind carbon dioxide, not oxygen CAM plants CAM plants plants that thrive in arid conditions and have their two stages of the Calvin cycle occurring at different times (separated by the vacuole) CO2 is converted into a C4 compound by PEP carboxylase during the night, when the stomata are open, and CO2 is able to diffuse into the leaf. The Calvin cycle that produces glucose occurs only during the day when stomata are closed. The C4 compounds are stored for breakdown during the day, where they will be broken down and CO2 is released. The stomata are closed and oxygen cannot be released but the concentration of CO2 should be higher so it can bind to Rubisco to form glucose through the steps of the Calvin cycle. Climate – Very hot, dry environments. E.g., cacti and agave Both stages take place in mesophyll cell  During the daytime, CAM plants do not open their stomata to prevent water loss Minimising photorespiration The carbon fixation stage takes place only at night when stomata are open. The Calvin cycle that produces glucose occurs only during the day when stomata are closed First stage- Stomata are Inorganic carbon dioxide from the air is fixed by the PEP carboxylase enzyme where there is no chance of binding oxygen, the products of this reaction are four-carbon organic acids, such as malic acid Second stage- occurs in mesophyll cells, but only in daylight when stomata are closed. the stored the malic acid is broken down in a reaction that releases carbon dioxide. The steady release of carbon dioxide creates a high concentration of CO2, an environment in which the Rubisco enzyme will preferentially bind carbon dioxide. As happens in the usual Calvin cycle, carbon dioxide is joined to an organic acceptor in a reaction catalysed by the Rubisco enzyme and enters the Calvin cycle. DOT POINT 6 - The factors that affect the rate of photosynthesis: light availability, water availability, temperature and carbon dioxide concentration The faster the rate of photosynthesis, the more glucose (and oxygen) that is produced in a shorter time frame LIGHT Photosynthesis cannot occur without light Plants use chlorophyll to absorb sunlight The photosynthetic rate increases as light intensity increases, until it reaches a maximal point When the graph plateaus the rate of reaction will no longer increase as the light saturation point has been reached light saturation point the point in which increasing the light intensity no longer increases the rate of photosynthesis Light colour greatest rate of photosynthesis occurs when a plant is exposed to violet or red light and that the rate of photosynthesis is relatively low under green light WATER As water availability increases, so does the rate of photosynthesis This rate will stop increasing when another factor becomes limiting If soils dry out and the water supply becomes too little, the rate of photosynthesis declines and then stops because closed stomata prevent the uptake of carbon dioxide needed for the Calvin cycle. If the water supply increases too much causing waterlogging of the soil, the rate of photosynthesis will also decline and stop because the lack of oxygen for cellular respiration in root cells stops water uptake. water deficit when there is a limited amount of water ○ plants close their stomata within minutes to prevent further water loss ○ uptake of CO2 is interrupted so that carbon dioxide levels within the leaves fall sharply ○ stops the Calvin cycle which stops photosynthesis waterlogged when excess water has reached a plant ○ not enough oxygen to enable plant root cells to respire adequately and gain the energy for living ○ no water supply to mesophyll cells of the leaves means that an essential input to the light-dependent stage of photosynthesis is not available and the rate of photosynthesis falls to zero TEMPERATURE above the optimal temperature, the enzymes begin to denature and are unable to function, causing a steep drop-off in photosynthesis rate and eventually the rate is zero as the enzymes which are vital can no longer be used As the ambient temperature is increased, the rate of photosynthesis also increases due to an increase in collisions between the reactants and the enzymes involved in photosynthesis. Eventually, as the heat passes a certain threshold, the enzymes start to denature, in which the tertiary structure of an enzyme is lost. This causes the rate to again decrease CARBON DIOXIDE C4 and CAM plants are less affected by carbon dioxide concentration reductions than C3 plants are As levels of carbon dioxide increase, so does the rate of photosynthesis until another factor - such as water, light or all chloroplasts operating at maximum efficiency - becomes limiting and the rate plateaus. Graph starts to plateau which could be due to the enzymes involved in carbon fixation working at maximum rate so that no further increase in rate is possible under the prevailing conditions Another possible reason is that the availability of essential coenzymes, such as NADPH, may have become a limiting factor.  Low CO2 levels make it more likely that O2 is bound and photorespiration occurs, thus removing an opportunity for photosynthesis and decreasing the overall photosynthetic rate ENZYME INHIBITION enzyme inhibitor a molecule that binds to and prevents an enzyme from functioning the presence of inhibitors lowers the rate of photosynthesis however, the effect of competitive reversible inhibitors can be gradually overcome if the substrate concentration is continually increased As enzyme inhibitors can target enzymes within any part of both stages of photosynthesis, all three types of plants are susceptible to the negative impact of inhibitors Increasing substrate concentration does not reduce the effect of irreversible inhibitors or reversible noncompetitive inhibitors. This means that the maximum possible rate of reaction is reduced in the presence of irreversible inhibitors or reversible non-competitive inhibitors. DOT POINT 7 - The main inputs, outputs and locations of glycolysis, Krebs cycle and electron transport chain including ATP yield (details of biochemical pathway mechanisms are not required) Cellular respiration process of converting chemical energy into a useable form by cells, typically ATP Two types of cellular respiration ○ aerobic cellular respiration cellular respiration that occurs in the presence of oxygen. Involves three stages, during which glucose and O2 are converted into ATP, CO2, and water ○ anaerobic fermentation a metabolic pathway that occurs in the absence of oxygen. Involves glycolysis, followed by further reactions that convert pyruvate  into lactic acid in animals, or ethanol and CO2 in yeast ○ use of ATP for living is continuous and, as a result, cellular respiration occurs all the time in every living cell MITOCHONDRIA mitochondrion (pl. mitochondria) a double-membrane-bound organelle that is the site of the second and third stages of aerobic cellular respiration includes an inner and outer membrane each composed of a phospholipid bilayer space inside the inner membrane is the mitochondrial matrix and is filled with a dense fluid containing many enzymes and solutes mitochondrial matrix the space inside the inner membrane of a mitochondrion. The site of the Krebs cycle inner membrane folds into peaks and ridges called cristae, which facilitate the function of the third stage of aerobic cellular respiration (the electron transport chain) crista (pl. cristae) the folds of the inner membrane of a mitochondrion. The site of the electron transport chain STAGES Glycolysis glycolysis the first stage of aerobic cellular respiration in which glucose is converted to two pyruvate molecules begins with the input of glucose, a six-carbon sugar molecule, and ends with two three-carbon molecules known as pyruvate ○ pyruvate a three-carbon molecule that can be formed from the breakdown of glucose via glycolysis Kreb’s cycle Krebs cycle second stage of aerobic respiration in which coenzymes are loaded and carbon dioxide is produced Pyruvate oxidation occurs before the Krebs cycle In an energy-releasing reaction, each pyruvate loses a C and an H atom (along with two oxygen), forming a 2C acetyl group that is delivered to the Krebs cycle by coenzyme A (as acetyl coenzyme A or acetyl-CoA) Electron transport chain Electron transport chain third stage of aerobic respiration in which there is a high yield of ATP can only operate if a supply of oxygen is available ?