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WEEK 1: DNA AND RNA DEOXYRIBONUCLEIC ACID (DNA) STRUCTURE ​ Deoxyribonucleic acid (DNA) HISTORY...

WEEK 1: DNA AND RNA DEOXYRIBONUCLEIC ACID (DNA) STRUCTURE ​ Deoxyribonucleic acid (DNA) HISTORY ○​ described as double helix by Watson and Crick ​ Molecular Biology ​ The helical structure of DNA results from the physicochemical ○​ coined by James Watson demands of the linear array of nucleotides. ○​ a field of science concerned with studying the chemical structures and processes ○​ a polymer – a long chainlike molecule made up of subunits called monomers of biological phenomena involving molecules ○​ name is a combination of: ○​ focusing on DNA,RNA, proteins and their interactions ​ Deoxyribo – a sugar ​ Molecular Diagnostics ​ Nucleic acid – a macromolecule made of nucleotides bound together ○​ a clinical application of molecular biology techniques to detect and diagnose by the phosphate and hydroxyl groups on their sugars diseases or predict disease risks ​ Nucleic acid chain grows by the attachment of the 5 ′ ​ Friedrich Meischer phosphate group of an incoming nucleotide to the 3 ′ ○​ published a paper on nuclein hydroxyl group of the last nucleotide on the growing chain ○​ isolated nuclein from the nucleus of WBC ○​ Anti-parallel - arrange of the DNA, which the 5' end of one strand of DNA ​ nuclein – was a viscous, nonprotein substance extracted from cell aligns with the 3' end of the other strand in a double helix. nuclei and was renamed DNA once the chemical composition of the ○​ We refer to DNA as oriented in a 5 ′ to 3 ′ direction, and the linear sequence of substance was discovered. the nucleotides, by convention, is read in that order. ​ Walther Flemming ○​ a macromolecule of carbon, nitrogen, oxygen, phosphorous and hydrogen atoms ○​ describing his work on the nucleus in 1882 (CHONP) ○​ admitted that the biological significance of the substance was unknown ○​ composed of 2 long strands made of building blocks called nucleotides bonded ​ James Watson and Francis Crick together ○​ first described the double helical structure of DNA in its hydrated form ​ Nucleotides – are linked together to form a polynucleotide. (B-form) ​ Nucleotide ​ It has 10.5 steps or pairs of nucleotides (base pairs [bp]) per turn ○​ basic unit of the DNA molecule ​ Rosalind Franklin ○​ 3 components of nucleotide: ○​ performed diffraction analyses about Watson and Crick’s molecular model as a ​ phosphate group physical evidence ​ deoxyribose sugar ​ CENTRAL DOGMA ​ nitrogen-containing base ○​ first proposed by Francis Crick ○​ Each nucleotide consists of five-carbon sugar ○​ describes the flow of genetic information from DNA to RNA to proteins ​ 1st carbon – covalently joined to a nitrogen base ○​ a theory that explains how genetic information flows from DNA to RNA, and ​ 5th carbon – covalently joined the phosphate group. then to protein. ○​ A nitrogen base bound to an unphosphorylated sugar is a nucleoside ○​ can be converted to nucleoside by hydrolysis ○​ Nucleotides are all identical except for their nitrogen base (A,T,G,C) MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 1  NUCLEOTIDE VS NUCLEOSIDE COMPONENTS OF NUCLEOTIDE NUCLEOTIDE NUCLEOSIDE 2' deoxyribose ​ pentose sugar Definition ​ A molecule consisting of a nitrogenous ​ composed of sugar and ​ particular type of pentose present in DNA base, a pentose sugar, and a nitrogenous base only; no ​ numbering of carbons should start at the carbon on the right, phosphate group. phosphate group which is after the oxygen molecule ​ Polymerizes to form DNA/RNA ​ cannot form directly ​ sugar molecule containing 5 carbons that has lost the hydroxyl ​ The individual phosphate groups are DNA/RNA (-OH) group on its 2' carbon. designated a, b, and γ, with the a ​ How does it differ from RNA sugar? phosphate being the one attached ○​ 2' deoxyribose – no oxygen attached on its 2nd carbon directly to the sugar ○​ Ribose – has OH- attached on its 2nd carbon Components Nitrogen base + Sugar + phosphate Nitrogen base + Sugar ​ The hydroxyl group on the 3rd carbon is important for forming Example 2’-deoxyadenosine 5’-triphosphate (dATP) Adenosine (A) the phosphodiester bond that is the backbone of the DNA strand. 2’-deoxyguanosine 5’-triphosphate (dGTP) Guanosine (G) Nitrogen bases ​ an alkaline cyclic molecule containing nitrogen 2’-deoxycytidine 5’-triphosphate 9 (dCTP) Cytidine (C) ​ planar carbon-nitrogen ring structures; the ladder of DNA 2’-deoxythymidine 5’-triphosphate (dTTP) Thymidine (T) ​ Example:: Function Building blocks of DNA & RNA, energy Precursor to nucleotides, ○​ Adenine → purine carriers (ATP, GTP), signaling molecules involved in metabolic pathway ○​ Thymine → pyrimidine (cAMP). ○​ Guanine → purine Solubility more soluble due to PO4 group Less soluble because no PO4 ○​ Cytosine → pyrimidine ​ Their differences in structure is due to amine and ketone ​ Polynucleotide substitutions, as well as the single or double bonds within the ○​ more than one nucleotides rings ○​ formed by attaching one nucleotide to another ○​ single-ring structure: pyrimidines through the phosphate groups. ○​ double-ring structure: purines ○​ phosphodiester bond – linkage between the ​ B-N-glycosidic bond nucleotides in a polynucleotide ○​ covalent bond that connects a nitrogenous base (purine ​ The nucleotide monomers are linked or pyrimidine) to the deoxyribose sugar in nucleotides together by joining a phosphate ○​ For purine: its Nitrogen 9 is attached to the Carbon 1 of 2' group, attached to the 5’ carbon of deoxyribose (N9–C1) one nucleotide, to the 3’ carbon of ○​ For pyrimidine: its Nitrogen 1 is attached to the Carbon 1 the next nucleotide in the chain. of 2' deoxyribose (N1–C1) ○​ Normally a polynucleotide is built up from ​ Hydrogen bond – links two nitrogen bases nucleoside triphosphate subunits ○​ An important feature of the polynucleotide is that the two ends of the molecule are not the same Phosphate group ​ Group of four oxygen atoms surrounding a central phosphorus atom found in the backbone of DNA MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 2  COMPLEMENTARY BASE PAIRING ​ The bases of DNA pair with each other in a predictable way. ○​ Adenine = Thymine ○​ Cytosine = Guanine ​ HYDROGEN PAIRING ○​ weak electrostatic attraction between an electronegative atom (such as oxygen or nitrogen) and a hydrogen atom attached to a second electronegative atom. ○​ The base pairing between adenine and thymine involves two hydrogen bonds, and that between guanine and cytosine involves three hydrogen bonds. ​ BASE PAIRING ○​ Because of the base pairing, the sequences of the two polynucleotides in the helix are complementary (the sequence of one polynucleotide determines the sequence of the other). ○​ Hybridization – the formation of hydrogen bonds between two complementary SUMMARY OF BONDS IN DNA strands of DNA Phosphodiester Bond β-N-Glycosidic Bond Hydrogen Bond Definition linking nucleotides between the nitrogen between together by connecting base and the sugar. complementary bases the sugar and phosphate group location Forms the Connects the Holds the two DNA sugar-phosphate nitrogenous base to the strands together backbone sugar in a nucleotide. between base pairs. DNA REPLICATION ​ Process by which DNA makes a copy of itself. Involves 3'-OH of one sugar and C1’ of sugar and N1 Nitrogenous bases (A-T, ​ It is semiconservative – the key to maintaining the sequence of the nucleotides in DNA 5'-phosphate of the next (pyrimidine) or N9 G-C) of opposite DNA through new generations nucleotide. (purine) of the base. strands ○​ meaning, when a DNA molecule is copied, each of the two resulting Bond Strong (Covalent) Strong (Covalent) Weak (Non-Covalent) double-stranded DNA molecules consists of one original (parental) strand and strength one newly synthesized strand. Function Provides structural Links the base to the Facilitates base pairing ​ As DNA synthesis proceeds in the 5 ′ to 3 ′ direction: integrity and sugar to form a and DNA double helix ○​ DNA polymerase – the enzyme responsible for polymerizing the nucleotide directionality (5’→3’) of nucleotide. stability. chains, uses a guide, or template → to determine which nucleotides to add to DNA. the chain Example between adjacent between adenine and A-T (2 hydrogen ​ Replication apparatus – designed to copy the DNA strands in an orderly way with nucleotides in a DNA deoxyribose in dATP bonds), G-C (3 minimal errors before each cell division strand. hydrogen bonds) MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 3  DNA REPLICATION PROCESS PROCESS ILLUSTRATION ENZYMES INvOLVE DESCRIPTION 1.​ INITIATION (1) Origin of Replication (ORI): -​ a specific DNA sequence/location where DNA replication begins (2) Formation of Replication Bubble: -​ At the ORI, the DNA double helix starts to unwind, forming a replication bubble. -​ The bubble has two replication forks that move in opposite directions as the DNA continues to unwind. 2.​ UNWINDING ​ Helicase (1)​ The replication bubble is further separated/unzipped into the 2 strands of DNA by the THE DNA ​ Gyrase Helicase (2)​ Helicase breaks the hydrogen bonds that hold the nucleotides together producing 2 template strands. (3)​ Gyrase assists helicase and reduces torsional strain and positive supercoil ahead of the replication fork. ○​ This is a type of topoisomerase that attached to parental DNA before it separated ○​ Located ahead of helicase 3.​ STABILIZING (1) SINGLE-STRAND BINDING (SSB) PROTEINS THE ○​ located after the helicase UNWOUND ○​ coat the separated strands/single-strand DNA to prevent them from rewinding into a DNA double helix ○​ prevent it from re-annealing or forming secondary structures during replication 4.​ PRIMING THE ​ Primase (1) The enzyme primase produces a short RNA primer (5-10 nucleotides) on each DNA strand. TEMPLATE ​ RNA Primers STRANDS ○​ short segments of RNA that allows the polymers to come and bind to start the replication process ○​ provides a starting point for DNA polymerase, as it cannot start synthesis from scratch 5.​ ELONGATION ​ DNA Polymerase III or (1)​ the RNA Primer is then attached by the DNA Polymerase III (in prokaryotes) or DNA DNA Polymerase δ/ε Polymerase δ/ε (in eukaryotes) ​ RNAse H or DNA (2)​ DNA POLYMERASE III → adds nucleotides with its 5′-phosphate group to the 3′ end of the Polymerase 1 last nucleotide of the primer. ​ DNA Ligase ○​ synthesis of the growing strand in the 5′-to-3′ direction involves adding nucleotides in a complementary order to the template strand. MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 4  ○​ both strands are read in the 3' to 5' direction ○​ add bases complementary to the template strand. (3)​ Synthesis of Leading Strand: ○​ DNA polymerase adds nucleotides continuously to the growing strand, same direction as the replication fork (4) Synthesis of Lagging Strand ○​ DNA polymerase synthesized discontinuously in the opposite direction of the replication fork → forming short fragments called "Okazaki Fragments” (5) Primer Removal & Gap Filling ○​ RNAse H (DNA polymerase I) → removes the RNA primer as DNA polymerase III approaches ​ an enzyme that hydrolyzes RNA from a complementary DNA strand, removes the primer RNA from the short RNA–DNA hybrid, and the resulting gap is filled by gap-filling DNA polymerase. ○​ then the DNA polymerase III will continue to move forward (6) Sealing the DNA Strand ○​ DNA ligase - joins Okazaki fragments on the lagging strand by forming phosphodiester bonds, creating a continuous strand ​ Replication Bubble forms at the origin of replication. ​ Helicase unwinds the DNA, creating replication forks at both ends of the bubble. ​ Primase adds RNA primers, and DNA polymerase adds new nucleotides to form new DNA strands. ​ On the leading strand, the new strand is built continuously, and on the lagging strand, it is built in small pieces (Okazaki fragments). ​ DNA ligase joins the fragments, completing the new DNA strands. ​ This entire process ensures that DNA is copied accurately before cell division MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 5  RECOMBINATION IN SEXUALLY REPRODUCING ORGANISMS RECOMBINATION IN ASEXUAL REPRODUCTION ​ Recombination ​ Horizontal Gene Transfer ○​ the mixture and assembly of new genetic combinations. ○​ refers to the movement of genetic material between organisms, other than ○​ occurs through the molecular process of crossing over or physical exchange through vertical transmission (from parent to offspring) between molecules ​ Recombinant Molecule Or Organism WAYS IN ASEXUAL REPRODUCTION ○​ one that holds a new combination of DNA sequences. Conjugation ​ involves two types of bacterial sexes: ​ The mixing of genes generates genetic diversity, increasing the opportunity for more ○​ F- → does not carry a genetic factor robust and well-adapted offspring. ○​ F+ → a had a “fertility factor” ​ Sexually reproducing organisms mix genes in three ways: ​ not only carried the information from one cell to another but also was responsible for establishing the WAYS IN SEXUAL REPRODUCTION physical connection between the mating bacteria 1 ​ At the beginning of meiosis, duplicated chromosomes line up ​ F factor – was shown to be an extrachromosomal ​ recombine by crossing over or breakage circle of dsDNA or plasmid carrying the genes coding ​ reunion of the four DNA duplexes for construction of the mating bridge. ​ This is a process where genetic material is directly transferred from 2 ​ The recombined duplexes are randomly assorted into gametes one bacterial cell to another through a physical connection called a ○​ so that each gamete contains one set of each of the recombined parental pilus. chromosomes. ​ It's akin to bacterial "mating" and often involves the transfer of 3 ​ The gamete will merge with a gamete from the other parent carrying its own set plasmids, which are small, circular DNA molecules separate from the of recombined chromosomes. bacterial chromosome PROCESS: (1)​ F− and F+ cells must be in contact with each other. (2)​ a filamentous bridge (pilus) is observed between mating bacteria (3)​ genetic information could move from F+ to F− bacteria but not from F − to F + bacteria (4)​ The fertility factor was transferred from F+ to F− bacteria in the mating process so that afterward, the F− bacteria became F+ MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 6  Transduction ​ Francois Jacob and Elie Wollman ○​ studied the transmission of units of heredity carried by viruses from one bacterium to another (transduction) ​ Alfred Hershey and Martha Chase ○​ confirmed that the DNA of a bacterial virus was the carrier of its genetic determination in the transduction process ​ this mechanism involves the transfer of genetic material from one bacterium to another via bacteriophages (viruses that infect bacteria). ○​ The phages inadvertently package bacterial DNA into their viral particles, which is then introduced to a new bacterial host. ​ Transduction is also useful in determining gene order Transformation ​ first observed in 1928 by Frederick Griffith ​ the basis for modern-day recombinant techniques ​ This process occurs when a bacterium takes up free, naked DNA fragments from its surrounding environment. ​ The DNA can come from dead and lysed bacteria, and once inside the cell, it can be incorporated into the bacterial genome. ​ What Griffith had observed was the transfer of DNA from one organism to another without the protection of a conjugative bridge or a viral coat. MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 7  RIBONUCLEIC ACID (RNA) ​ In Eukaryotes, rRNA is synthesized from highly repeated gene ​ polymer of nucleotides similar to DNA. clusters ​ differs from DNA in a way that: o​ copied from DNA as a single 45S precursor RNA ○​ having ribose instead of deoxyribose (pre-ribosomal RNA) that is subsequently processed into: ○​ having uracil instead of thymine ○​ single strand rather than as a double helix. ▪​ 18S species of the ribosome small subunit ​ although they do not have complementary partner strands, they are not completely ▪​ 5.8S and 28S species of the large subunit single strand ▪​ 5S found in the largest subunit ​ RNA can also pair with complementary single strands of DNA or another RNA and form ​ Another subunit 5S in eukaryotes synthesized separately a double helix TRANSFER RNA ​ Facilitates the translation of information from nucleic acid to (tRNA) protein requires reading of the mRNA by ribosomes, using adaptor TYPES OF RNA molecules. MESSENGER ​ initial connection between the information stored in DNA and the ​ Synthesized in the nucleolus RNA (mRNA) translation apparatus that will ultimately produce the protein ​ relatively short, single-stranded polynucleotides of 73 to 93 bases products responsible for the phenotype. in length, MW 24,000 to 31,000. ​ Prokaryotic mRNA is sometimes polycistronic ​ There is at least one tRNA for each amino acid o​ one mRNA codes for more than one protein. ​ cruciform structure of four to five double-stranded stems and ​ Eukaryotic mRNA, in contrast, is monocistronic, three to four single-stranded loop o​ having only one protein per mRNA o​ Undergoes a series of transcriptional events before it is TRANSCRIPTION PROCESS translated into proteins ​ Transcription ​ Amount of particular mRNA in a cell is related to the requirement o​ DNA to mRNA occurs mostly in interphase; in the nucleus(eukaryotes) and for its final product cytoplasm (eukaryotes) o​ Constitutive transcription – transcribe constantly in the cell 3 PHASES OF RNA PROCESSING o​ Inducible/Regulatory Transcription – transcribe only at INITIATION RNA polymerase and its supporting accessory certain times during mitosis proteins assemble on DNA at a specific site called RIBOSOMAL ​ largest component of cellular RNA the promoter. RNA (rRNA) o​ comprising 80% to 90% of the total cellular RNA ​ Promoter region- DNA sequence that can be ​ an important structural and functional part of the ribosomes, recognized by transcription factors cellular organelles where proteins are synthesized – Rough ​ Transcription factors- that bind specific Endoplasmic Reticulum sequencs in the DNA; bind to the pro moter ​ Prokaryotes has 3 RNA species: (all synthesized from the same region( will form initiation complex) gene) ​ Initiation complex- attracts RNA polymerase o​ 16S – found in the ribosome small subunit ​ Coding region- part that will actually be transcribed o​ 23S and 5S – found in the ribosome large subunit MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 8  ​ Activator or repressor region- controls the ​ In order for splicing to occur we need to level of transcription form something called spliceosome ​ Enhancer region (consisting of snRNA)small nuclear RNA ELONGATION 1.​ RNA polymerases in both eukaryotes and may then join with some proteins to form prokaryotes synthesize RNA using the base snRNPs(part of spliceosome which allows sequence of one strand of the double helix us to cleave introns and join exons as a guide. together. 2.​ The DNA double helix is locally unwound ​ snRPS will bind near the end of the exons into single strands to allow the assembly (snurps will join together and form a and passage of the transcription machinery, spliceosome) forming a transcription bubble. ​ splicing happens in the nucleus 3.​ The RNA will copy the DNA template with ​ snRPS join together and creates a loop and the introns is cleaved and degraded complementary bases same with DNA but 1.​ on the 5’ end we need to add a CAP uracil instead of thymine and the process ​ 3 biomolecules that prevents degradation from exonucleases; protects our continues. mRNA from being degraded 4.​ RNA synthesis does not require priming 2.​ On the 3’ end we added a poly A tail; group of adenine nucleotide; repent TERMINATION ​ There is a termination sequence that will notify degradation that that’s the end of the coding region ​ After splicing and addition of CAP and poly A tail, mRNA is capable of leaving ​ Termination sequence- point where transcription the nucleus stops ​ And transfer into the cytoplasm in order to be translated and converted into ​ When RNA polymerase reaches the termination proteins sequence RNA polymerase is released from the template strand ​ Rho proteins sometimes bind to termination sequence and can facilitate the process of RNA pol. Leaving ​ When that happens the primary transcript will be ready for processing of introns and exons and capping and adding of poly A tail PROCESSING(PRE-MRNA) Before the pre-mRNA exits the nucleus, it has to undergo a series of processing ​ Splicing- separating exons and introns and removing all the intron segments from our primary transcript. ​ INTRONS- are not translated into proteins ​ EXONS- are the ones that will be translated ( exons will EXIT the nucleus to be translated and expressed MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 9  TOPIC 2 : PROTEINS AMINO ACIDS ​ Building blocks of protein, monomer units of the polypeptide chains PROTEINS ​ has characteristic biochemical properties determined by the nature of its amino acid side ​ the most abundant macromolecules in cell chain ​ products of transcription and translation of the nucleic acids ​ grouped according to their polarity → tendency to interact with water at pH. ​ Manifestation of the ultimate effect of the information stored and delivered by the ​ nonpolar, uncharged polar, negatively charged polar, and positively charged polar nucleic acid ​ manifest the phenotype (observable characteristics) directed by the nucleic acid Classifications of Amino Acids Based on Polarity of their Side Chains information CLASSIFICATION AMINO ACID ABBREVIATIONS ​ Found throughout the body (skin, muscle, bone, hair etc.) and has specific functions- like Nonpolar Alanine Ala, A hemoglobin, enzymes Isoleucine Ile, I ​ Proteome - the collection of proteins encoded in all of an organism’s DNA Leucine Leu, L ​ The information stored in the sequence of nucleotides in DNA is transcribed and translated into an amino acid sequence that will ultimately bring about the genetically Methionine Met, M coded phenotype. Phenylalanine Phe, P ​ The proteome of humans is larger than the genome - a single gene can give rise to Tryptophan Trp, W more than one protein through alternate splicing and other post-transcriptional/ Valine Val, V post-translational modifications.’ Polar Asparagine Asn, N ​ Proteins are classified according to function as: Cysteine Cys, C a.​ Enzymes * Glutamine Gln, Q b.​ Transport * Glycine Gly, G c.​ Storage Proline Pro, P d.​ Motility^ Serine Ser, S e.​ Structural ^ Threonine Thr, T f.​ Defense* Tyrosine Tyr, Y g.​ Regulatory Protein* Negative Charged (acidic) Aspartic Acid Asp, D -​ * are usually globular in nature (soluble , diffuse freely across membranes) Glutamic Acid Glu, E -​ ^ are fibrous and insoluble Positively Charged (basic) Arginine Arg, R ​ Conjugated proteins Histidine Hid, H ○​ do have components other than amino acids. Lysine Lys, K ○​ prosthetic group - the nonprotein component of a conjugated protein is called ○​ Examples of conjugated protein: ​ Hemoglobin ​ lipoprotein (LDL, HDL) ​ glycoprotein MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 10 ​ There are 20 common amino acids (AA) and they are divided into: ○​ Essential – AA in which the body cannot be produced on its own (diet and supplements) ○​ Non-essential – NOT required by the body but can contribute to overall health ○​ Conditionally essential – important in times of illness and stress ​ There are two non-canonical amino acids: unnatural, found in organisms or made in laboratory: ○​ Pyrrolysine is found in Archaebacteria (coded by UAG ○​ Selenocysteine in Selenoproteins, (glutathione peroxidase and formate dehydrogenase, Coded by UGA) ​ Changes in translation of UGA can lead to symptoms of selenium deficiency ​ properties of amino acids that make up a protein determine the shape and biochemical nature of the protein synthesized in vivo by stereospecific enzymes so that naturally occurring proteins are made of amino acids of L-stereochemistry (not D-stereochemistry) ​ The central asymmetric carbon atom of the amino acid is attached to: ○​ carboxyl group ○​ amino group ○​ hydrogen atom ○​ side chain ​ At pH 7, most of the carboxyl groups of the amino acids are ionized ​ Proline - side chain is cyclic, with the amino group attached to the end carbon of the ​ Zwitterions side chain making a five-carbon ring (differ from the rest) ○​ simultaneously electrically charged and electrically neutral and contain positive ​ Two AA joined together by a peptide bond make a dipeptide , three (tripeptide) etc. and negative charges, but the net charge on the molecule is zero. ​ At one end of the peptide will be an: ○​ The ionization can switch between the amino and carboxyl groups ○​ amino group – the amino-terminal, or NH 2 end ​ an internal transfer of a hydrogen ion from the -COOH group (donor) to ○​ carboxyl group – at the opposite terminus of the peptide, the carboxy-terminal, the -NH2 (acceptor) or COOH end. ​ group to leave an ion with both a negative charge and a positive charge ​ peptide chains grow from the amino to the carboxy terminus. ​ pK value - pH levels where the AA will become completely positively or negatively ​ also classified by their biosynthetic origins or similar structures based on a common charged. biosynthetic precursor. ​ pI value - pH where an AA is neutral, its positive and negative charges are in balance, ​ Histidine – has a unique synthetic pathway using metabolites common to purine determined by the ionization state of the side chains of its constituent amino acids. nucleotide biosynthesis, which affords the connection of amino acid synthesis to ​ Peptide bond – form protein backbone, the amino and carboxyl terminal groups of the nucleotide synthesis amino acids are joined in a carbon-carbon-nitrogen (–C–C–N–) substituted amide linkage MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 11  SEQUENCE OF AMINO ACIDS Tertiary Structure ​ connected by disulfide bonds (S2) ​ Sequence of amino acids in a protein determines the nature and activity of the protein. ​ happens when secondary structures assemble into larger functional units that include mature polypeptides and its Primary Structure ○​ The sequence of amino acids in a polypeptide chain read by component domains convention from the amino terminal end to the carboxy terminal ​ Important for protein function end ​ If a protein is DENATURED – it loses its tertiary structure and it ○​ Minor changes in primary structure can alter the activity of becomes improperly coiled and non-functional proteins dramatically. ​ It is affected also by mutations ○​ The single amino acid substitution that produces hemoglobin S in ​ Prions – infectious acellular microbes that can change the tertiary sickle cell anemia structure, can causes ○​ Minor changes in primary structures can have such drastic ​ Creutzfeldt–Jakob disease effects because the amino acids must often cooperate with one ​ bovine spongiform encephalitis (mad cow disease) another to bring about protein structure and function Quaternary ​ Involves the number and types of polypeptide units of oligomeric Structure proteins and their spatial arrangement Secondary ​ Folding of short contiguous segments of polypeptide into ​ Oligomers - are proteins that bound together to function form a Structure predictable configurations from interactions between amino acid dimer (two), trimer (three), and tetramer (four). side chains. ​ Two configurations: First describe by Linus Pauling and Robert Corey GENE ○​ The alpha-helix – random coils, less ordered ​ defined as the ordered sequence of nucleotides on a chromosome that encodes a ○​ The beta-pleated – ordered beta specific functional product ​ H-bond – connects C and NH terminals in alpha helices ​ Gene contains: ​ Specialized Secondary Structures: ○​ Structural sequence - code for an amino acid sequence ○​ Zinc finger - frequently found in proteins that bind to Dna ○​ Regulatory sequence - important for the regulated expression of the gene ○​ Leucine Zipper - also found in transcription factors ​ It is the fundamental physical and functional unit of inheritance ​ Part of chromosome responsible for the phenotype affected by mutations ​ Not delineated well in terms of their physical size but were mapped relative to each other based on the frequency of recombination between them MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 12  GENETIC CODE TRANSLATION ​ The language that codes the amino acids, ​ occurs in the cytoplasm, specifically within the ribosomes ​ It is said to be universal ​ it involves the synthesis of proteins by translating the sequence of nucleotides in ​ Codons - A sequence of three consecutive nucleotides in a DNA or RNA molecule that mRNA to a sequence of amino acids codes for a specific amino acid but the genetic code is written in RNA language ​ the mRNA leaves the nucleus and binds to the ribosome ​ The code is redundant, such that amino acids have more than one codon ​ Eukaryotic cells – can contain up to 10 million ribosomes ​ Methionine and tryptophan – amino acids with only one codon ​ Bacterial cells – may have over 70,000 ribosomes, depending on growth rate. ​ Nonsense codons – codons that terminate synthesis ​ tRNA ​ Stop codons include: ○​ a type of RNA that can recognize components of both nucleic acid and protein ○​ UAG sequences ○​ UAA ○​ carries three-base anticodon, complementary to the codon of a specific AA ○​ UGA ○​ Protein synthesis starts with activation of the amino acids by covalent ​ A total of 64 different codons – 61 for synthesis and 3 stop codons attachment to tRNA, or tRNA or tRNA charging ​ Characteristics of the genetic code have consequences for molecular analysis. ​ Aminoacyl tRNA synthetases – enzyme that catalyzes the reaction, ​ Mutations will have different effects on phenotype (depends on the resultant changes in dependent on Mg2+ the AA sequence) ​ Two steps: ○​ mutations range from phenotypically silent to drastic ○​ (1) the amino acid is activated by the addition of AMP ​ amino acid + ATP → aminoacyl-AMP + PPi ○​ (2) the activated amino acid is joined to the tRNA ​ aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP ​ The product of the reaction is an ester bond between the 3 ′ hydroxyl of the terminal adenine of the tRNA and the carboxyl group of the amino acid ​ there are 20 amino acid tRNA synthetases ○​ one for each amino acid ○​ Designated as class I and class II synthetases ​ these enzymes interact, respectively, with the minor or major groove of the tRNA acceptor arm. ​ Both classes also recognize tRNAs by their anticodon sequences and amino acids by their side chains and only the appropriate tRNA and amino acid will fit into its cognate synthetase. ​ An errant amino acid bound to the wrong synthetase will dissociate rapidly before any conformation changes and charging can occur MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 13  PROTEIN SYNTHESIS ​ The resulting functional 70S or 80S ribosome is the initiation complex P site –The initiator tRNA (tRNA-Met or tRNA-fMet) binds at the P site, while other tRNAs later tRNA CHARGING bind at the A site. ELONGATION ​ Elongation - tRNA carrying the next amino acid binds to the A site of the ribosome in a complex with elongation factor Tu (EF-Tu) and GTP ​ The fit of the incoming tRNA takes place by recognition and then proofreading of the codon anticodon base pairing ​ Hydrolysis of GTP by EF-Tu occurs between these two steps: (1)​ The EF-Tu-GDP is released INITIATION (2)​ The EF-Tu-GTP is regenerated by another elongation factor, EF-Ts ​ Protein synthesis in the ribosome almost always starts with: ​ The first peptide bond forms between the amino acids at the A site and P site. ○​ Methionine – in eukaryotes ​ The N-formylmethionyl (fMet) group of the first amino acid is transferred to the amino ○​ N-formylmethionine – bacteria, mitochondria, and chloroplasts group of the second amino acid, generating a dipeptidyl-tRNA in the A site. ​ Initiating factors that participate in the formation of the ribosome complex differentiate ​ This reaction is catalyzed by peptidyl transferase, an enzymatic function of the large the initiating methionyl tRNAs from those that add methionine internally to the protein ribosomal subunit. ○​ Initiation factor 3 (IF-3) –where the small ribosomal unit binds first ​ Peptidyl transferase activity is mediated by rRNA (ribozymes), though nearby ​ Ribosomal binding site – these are specific sequences near the 5 ′ end of the mRNA ribosomal proteins may influence the reaction’s efficiency. that the ribosomal subunit will bind after ​ Wobble base pairing allows flexibility at the third codon position. ​ This guides the AUG codon (the “start” codon) to the proper place in the ribosomal subunit Translocation of tRNAs (Ribosome Movement & Energy Use) ​ Initiating Factor 2 (IF-2) - binds to GTP and the initiating tRNA Met or tRNA fMet , then join the complex. ​ Peptidyl transferase – catalyzes the peptide bond formation from the large sub-unit ​ The large ribosomal subunit then associates with the hydrolysis of GTP and release of ​ The ribosome moves, shifting the dipeptidyl-tRNA from the A site to the P site with GDP and phosphate, IF-2, and IF-3. the release of the “empty” tRNA from a third position, the E site, of the ribosome ​ The resulting functional 70S or 80S ribosome is the initiation complex ​ Distance of movement: ​ P site – peptidyl site of the functional ribosome where the tRNA Met or tRNA fMet (can -​ A → P site: 20 angstroms only bind in P site) is situated -​ P → E site: 28 angstroms ​ all other tRNAs bind to an adjacent site,Initiating Factor 2 (IF-2) - binds to GTP and ​ Elongation Factor EF-G – required for the movement the initiating tRNA Met or tRNA fMet , then joins the complex. ​ As the ribosomal complex moves along the mRNA, the growing peptide chain is always ​ The large ribosomal subunit then associates with the hydrolysis of GTP and release of attached to the incoming amino acid GDP and phosphate, IF-2, and IF-3. ​ Two GTPs are hydrolyzed to GDP with the addition of each amino acid (GTP hydrolysis, which provides energy.) MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 14 ​ Each amino acid addition requires hydrolysis of 2 GTP molecules. TERMINATION ​ The ribosomal subunits undergo shifting and rotation as they move along t ​ Termination occurs when the ribosome encounters a stop codon ​ Chaperones – specialized proteins that assist the folding of the growing peptides into ○​ UAA, UAG, or UGA (nonsense codons). their mature configuration ○​ Stop codons do not code for any amino acid and are recognized by release Protein Folding During Translation factors (RFs). ​ When the ribosome encounters a termination codon: ​ As the polypeptide grows, it begins to fold into its functional 3D shape. ○​ Prokaryotes: termination, or release factors (R 1 , R 2 , and S in E. coli ), ​ Molecular chaperones assist in proper folding by: ○​ Release Factors (RFs) Involved: -​ Binding to the large ribosomal subunit. a.​ RF1 recognizes UAA & UAG. -​ Creating a hydrophobic pocket that protects the growing peptide/ holds b.​ RF2 recognizes UAA & UGA. the emerging polypeptide c.​ RF3 aids in release factor recycling. ​ Without chaperones, unfinished proteins could misfold or form nonfunctional ​ The release factors trigger hydrolysis of the peptidyl-tRNA bond, freeing the finished aggregates. protein. ​ Folding occurs co-translationally, meaning the protein adopts its structure while still ​ The ribosomal subunits dissociate, allowing for a new round of translation. being synthesized. ○​ Eukaryotes: termination codon–mediated binding of polypeptide chain release factors (eRF1 and eRF3) triggers hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center ○​ eRF1 recognizes all three stop codons. ○​ eRF3 assists in GTP hydrolysis and peptide release. ○​ Hydrolysis occurs at the peptidyl transferase center in the large subunit. MLS 123: MOLECULAR BIOLOGY​ | EC, JPT, MSCY​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 15