Introduction to Genetics PDF

Nutritional Biochemistry Introduction to genetics DIET413/BHCS1019 Dr Nathaniel Clark FHEA RNutr MRSB [email protected] 1 Questions 1. Proteins that span the membrane with alpha 1. Hydrophilic. helices contain which k...

Nutritional Biochemistry Introduction to genetics DIET413/BHCS1019 Dr Nathaniel Clark FHEA RNutr MRSB [email protected] 1 Questions 1. Proteins that span the membrane with alpha 1. Hydrophilic. helices contain which kind of side chains in the 2. Channel proteins merely allow solutes to flow down their concentration gradient, amino acids that are central to this complex? rather than picking them up and 2. The transport proteins that move solutes transporting them against their gradient. against a concentration gradient are all carrier 3. In simple diffusion, the substance in proteins, rather than channel proteins. Why? question can diffuse down its concentration gradient through the 3. Differentiate between simple diffusion and membrane. In facilitated diffusion, the facilitated diffusion. substance is not lipophilic and cannot directly diffuse through the membrane. A 4. What are the two forms of energy that can channel or carrier is required to facilitate power active transport? movement down the gradient. 5. The K+ channel and the Na+ channel have 4. The two forms are: (i) ATP hydrolysis and (ii) the movement of one molecule down similar structures and are arranged in the same its concentration gradient coupled with the orientation in the cell membrane. Yet the Na+ movement of another molecule up its channel allows sodium ions to flow into the cell concentration gradient. and the K+ channel allows potassium ions to 5. An ion channel must transport ions in flow out of the cell. Explain. either direction at the same rate. The net flow of ions is determined only by the composition of the solutions on either side. 2 Learning outcomes 1. Describe the structure of DNA and how it is organised in the nucleus. 2. Describe the process of DNA replication. 3. Outline the transcription and translation of a gene into a protein. 4. Briefly outline the structure, control and role of mtDNA, and linked diseases. 3 1. Genes and genetics 4 1. The genome Every cell in an organism contains essentially the same genetic information in the genome. The genome controls the biochemical activity of the cell in response to multiple stimuli. It is also the genome which contains the inherited traits which are transmitted from generation to generation. Reductionist. The sequencing of the human genome was completed in 2003. The human genome contains around 20,000 to 21,000 genes. 99.9% of these are the same between humans. 0.1% are the factors for disease. Individual genes control the synthesis of specific proteins. 5 1. The structure of DNA The genome is made of DNA. DNA is a polymer consisting of a long chain of monomers called nucleotides. Thus the DNA molecule is said to be a polynucleotide. DNA has a helical structure that is analogous to a ladder. 2 nm wide. 10.5 “steps” per turn (=3.4 nm). How does it twist compared to alpha helix? 6 1. DNA methylation 7 1. Nucleosides Consist of two parts: a sugar and a nitrogen containing ring. The sugar plus the base are called a nucleoside. The bases are attached to the sugar molecule by a bond between the (anomeric) 1ʹ carbon of the sugar and a Adenin e Thymin e nitrogen at position 9 of the purines or position 1 of the Guanin Cytosin pyrimidines. e e 8 1. Nucleotide structure These form the “steps”. Each nucleotide has 3 parts: a sugar, a nitrogen containing ring structure called a base, and a phosphate group. Nucleotides contain a phosphate group [PO4] attached to the 5ʹ carbon of the sugar. The sugar is a 5 carbon pentose DNA called 2ʹ-deoxyribose in which the –OH group on carbon 2 is replaced by hydrogen. 9 1. Hydrogen bonding between bases Bases only bond to specific bases: Adenine only bonds to Thymine. Guanine only bonds to Cytosine. Complementary base pairing. 2 hydrogen bonds form between A and T, and 3 hydrogen bonds form between C and G. This concept of base pairing is the basis of the mechanisms of: DNA replication. Transcription. Translation. 10 1. Polynucleotides Nucleotides join to form polynucleotides which are the structural units of DNA. The 5ʹ phosphate of one nucleotide forms a bond with the 3ʹ carbon of the next nucleotide (eliminating the OH group of the 3ʹ carbon in the process). Important for enzyme recognition. The bond is called a 3ʹ-5ʹ phosphodiester bond. The phosphodiester bonding allows the formation of a long chain of polynucleotides – phosphate-sugar “backbone”. 11 1. The DNA double helix The relationship between the deoxyribose chains and the binding between the bases and the sugars causes the structure of the DNA molecule to take the shape of a double helix. In one deoxyribose chain the oxygen of the ribose is above the carbons (on the left). In the other chain the oxygen is below the carbons Therefore, the strands are antiparallel i.e. they run in opposite directions - 3’ and 5’. This concept of directionality of nucleic acid strands is essential to understanding the mechanisms of replication and transcription. 5’ contains the phosphate. 12 1. Histones, chromatin and chromosomes DNA in cells is compacted for storage. DNA is first wrapped around specific proteins called histones (8 proteins), forming nucleosomes. Histones are chemically modified by enzymes to regulate gene transcription (epigenetics: DIET414/BHCS1020). The nucleosomes are condensed into a chromatin fibre. Finally the chromatin is arranged into chromosomes. https://www.youtube.com/watch?v=gbSIBh FwQ4s 13 1. Structure of genes A gene is a discrete segment of DNA that encodes the amino acid sequence of a polypeptide. The basic unit of inheritance as they give rise to physical characteristics. They are found on the chromosomes in bands. The end of chromosomes are capped by telomeres. These are repetitive DNA elements that protect the ends of the chromosomes from degradation, and from end to end fusion with other chromosomes. 14 Activity With the person next to you, explain how the ends of a DNA sequence are chemically distinct. How does this relate to the structure of the DNA? To aid revision: make a list of key words and their associated definitions to be able to describe the structure of DNA. Then do the same but for Learning outcome 2 (DNA replication). 15 2. The cell cycle DNA replication forms part of the cell cycle. A well organised sequence of events encompassing the time a cell takes to divide into 2 daughter cells. The events of the cycle include G1 where there is an increase in the amount of cytoplasm and an increase in the number of organelles. The precise duplication of DNA occurs in the S phase. Further growth of the cell and cellular organelles occurs in the G2 phase. Mitosis and cell division occurs in the M phase. 16 2. The cell cycle and the checkpoints 17 2. DNA replication To provide a full copy of DNA in the two daughter cells, the single copy of DNA must be replicated to provide two identical copies (one for each daughter cell). This process of copying the DNA is called replication. Each chromosome replicates its DNA by unwinding the double helix and synthesising a complimentary daughter strand on each parent strand. 18 2. DNA replication - unzipping The separation of the double helix is achieved by the action of a helicase enzyme. Unzipping of DNA occurs at distinct positions called the replication origin that contain sequences rich in weaker A-T base pairs, and usually progresses in both directions. Why are they weaker? Then DNA polymerase synthesises DNA. DNA polymerases require a short double stranded region to initiate DNA synthesis (a primer). This is produced by an RNA polymerase called primase. The primase synthesises a short RNA primer sequence on the DNA template creating a short double stranded region. 19 2. DNA replication – the replication fork The region where the DNA unwinds is called the replication fork. The synthesis has leading and lagging strands. Synthesis of DNA by DNA polymerases occurs only in the 5ʹ → 3ʹ direction. As the two strands of DNA run in opposite directions (one runs 5ʹ→3ʹ, the other 3ʹ→5ʹ) slightly different mechanisms are required to synthesise each. 20 2. DNA replication – leading/lagging strands One strand, the leading strand, is copied in the same direction as the unwinding helix. The other strand, the lagging strand is synthesised in the opposite direction to the growing replication fork and is copied discontinuously. The fork is undoing the further away from the origin. The lagging strand is synthesised in a series of fragments called Okazaki fragments. 21 2. DNA replication – lagging strand On the lagging strand, synthesis ends when the next RNA primer is encountered. At this point a different DNA polymerase (I) takes over and removes the RNA primer replacing it with DNA. The final stage required to complete synthesis of the lagging strand is for the Okazaki fragments to be joined together by phosphodiester bonds. This is carried out by a DNA ligase enzyme. The base pairing ensures that the daughter strand formed by the action of DNA polymerase is a complementary strand to the parent template. Thus the two new DNA strands consist of one strand of old DNA linked to a new strand of complementary DNA. 22 2. DNA replication - storage Once DNA has been replicated it needs to be in a form suitable so that one copy can go into one of the new cells and one into the other. When the cell approaches mitosis the DNA is arranged in a form suitable for rearrangement into two daughter cells. This form of DNA is referred to as chromosomes. https://www.youtube.com/watc h?v=TNKWgcFPHqw 23 10 min break 24 3. From genes to proteins Each DNA molecule is made up of a large numbers of genes. Each gene is therefore a small section of the entire DNA molecule. The main function of genes is to direct the synthesis of specific proteins within cells accurately and at the right time. This process is normally regulated by hormones, growth factors, etc. The processes involved in gene regulated protein synthesis are: 1. Transcription. 2. Translation. 25 3. Transcription The genes, through their sequence of bases, contain the information which allows the correct sequence of amino acids to be assembled to make up a specific protein molecule. Thus, to provide this information to the protein synthesis machinery, the code must be transcribed into a message, and this must be carried to the ribosome. This process is transcription. The messenger is another nucleic acid called messenger RNA (mRNA). Single stranded and uracil replaces thymine. 26 3. Initiation of transcription - TFs In mammalian cells the start point for transcription is a complex of proteins associated with DNA called general transcription factors. They are associated with the promoter region of DNA where transcription starts and help control the rate of transcription of the gene. The complex of proteins that make up the transcription factors include binding sites for hormones and messengers which turn on specific genes. 27 3. Initiation of transcription – other regions In addition to the general transcription factors are other DNA elements called enhancers which bind to the transcription factors and greatly enhance the activation of the gene (via protein binding). These enhancers can be some distance away from the general transcription factor complex. The TATA box (a promoter) is located about 25-30 base pairs upstream of the transcription initiation site in 24% of genes. The function of the TATA box is to locate the RNA polymerase at the correct site to initiate transcription. 28 3. Chain initiation The RNA polymerase enzyme is responsible for assembling the bases in the RNA molecule. After the initial binding step, RNA polymerase initiates RNA synthesis at the transcription start site. It places the first nucleotide at this site and the sequence of nucleotides is then added according to the sequence on DNA. 29 3. Chain elongation The bases are the same as in DNA except that uracil replaces thymine in the RNA molecule. Only one strand of DNA acts as the template for RNA synthesis. RNA polymerase moves from the 3’ end of the template strand, creating an RNA strand that grows in a 5’ to 3’ direction. Opposite to DNA replication. 30 3. Chain termination Special base sequences also terminate RNA synthesis. When the RNA polymerase reaches the transcription- termination sequence on DNA, the polymerase enzyme dissociates from DNA and the newly synthesised RNA molecule is released. https://www.youtube.com/watch? v=WsofH466lqk 31 3. Post-transcriptional modification The DNA molecule which is used as a template for RNA synthesis contains regions which code for proteins called exons and sections which do not code for proteins called introns. In the initial transcript these sections are included in the RNA molecule. The introns are removed from the RNA molecule by a process called mRNA splicing to leave just the protein coding sequences. 32 3. Translation The next process is the attaching of amino acids in the sequence coded by the mRNA to synthesise the protein. This requires two assistants: ribosomes and transfer (tRNA). Ribosomes are large protein molecules formed by the incorporation of proteins with ribosomal RNA (rRNA). Ribosomes consist of two subunits a large and a small subunit. The subunits remain separate in the cytosol and do not combine until they are participating in protein synthesis. 33 3. Ribosomes Ribosomes are the site of protein synthesis. The mRNA passes through the ribosome and amino acids are added according to the triplet code (codon; 3 base pairs per amino acid) sequence contained on the mRNA. tRNA exists in the cytosol and has a ‘clover leaf’ type structure. The two most important sites are: 1. The amino acid binding site. 2. The triplet sequence making up the anticodon. 34 3. Transfer RNA There are a number of tRNA molecules in the cell, each of which can carry a specific amino acid and have a specific triplet anticodon. The anticodon binds to the complementary triplet code on the mRNA so that it brings the amino acids to the mRNA in the correct sequence. The ribosome has two binding sites for tRNA the A site and the P site. The first tRNA carrying the start anticodon (UAC) then binds to either the A or P site adjacent to the codon AUG which is the start codon for protein synthesis. 35 3. The triplet code 36 3. Elongation peptide chain Once the start codon is occupied, translation of next codons on the mRNA begins. The following amino acids specified by the mRNA codons are then joined together in sequence to form the polypeptide chain. The second tRNA molecule binds to the A or P site not occupied by the starting tRNA. The adjacent amino acids are then joined together by peptide bonds. The joining of amino acids to form the peptide is believed to be mediated by the enzyme peptidyl transferase. Once the reaction has occurred the amino acid leaves the tRNA which dissociates and returns to the cytosol to collect another amino acid. 37 3. Termination The addition of amino acids continues until the presence of a stop codon on the mRNA is reached. At this point the synthesis is complete and the completed polypeptide is released. The ribosome then dissociates from the mRNA. https://www.youtube.com/watch? v=5bLEDd-PSTQ 38 4. Mitochondrial DNA (mtDNA) Mitochondria produce the majority of cellular ATP by oxidative phosphorylation. Contain important biochemical pathways e.g the tricarboxylic acid cycle (TCA) and part of the urea cycle. It is also important in the regulation of apoptosis and in regulating cytosolic Ca2+ concentration. Mitochondria contain the only extranuclear source of DNA in animal cells. Mitochondrial DNA is a circular double stranded 16,569 base pair molecule of DNA. 39 4. Mitochondrial DNA mtDNA encodes 37 genes including: 13 essential polypeptides for oxidative phosphorylation. 2 ribosomal RNAs. 22 transfer RNAs. The remaining proteins needed for oxidative phosphorylation are synthesised in the cytosol and transported to the mitochondria. Only 2% is junk DNA (331 nucleotides; compared to 98% junk in nDNA, 294 billion). 40 4. mtDNA replication Inheritance through the maternal line. The method of mtDNA replication is controversial: It is basically similar to that occurring in nuclear DNA. The controversy concerns the timing of the production of the lagging strand. There are 2 theories: 1. Strand-asymmetric model suggests the leading strand is 66% complete before the lagging strand replication starts. 2. Strand-coupled model argues that the leading and lagging strands are made at the same time. 41 4. Transcription and replication The basal machinery for transcription initiation of mtDNA is similar to that of nuclear DNA. It consists of a set of 3 proteins: 1. Mitochondrial RNA polymerase. 2. A helicase called twinkle helicase to unwind DNA. 3. A mitochondrial single-stranded DNA binding protein. 42 4. Transcription and replication - differences There are no introns, and there are either no or very few non-coding bases between adjacent genes. Generally there are no termination codons in the gene itself. These are created post-transcriptionally by polyadenation. 43 4. Mitochondrial disease Mitochondrial dysfunction is increasingly being recognised as a major contributing factor to ageing and age related degenerative diseases. mtDNA is at risk of damage caused by free radicals (reactive oxygen species). Because mtDNA has multiple copies, damage to one copy does not lead to cellular dysfunction. In order to have an influence on cellular function the number of copies damaged needs to reach a threshold level (usually 50-70%). 44 4. Energy failure through damage Cells that cannot produce enough ATP aerobically i.e. by oxidative phosphorylation may shunt pyruvate to lactate in an attempt to produce more ATP anaerobically. This leads to cellular acidosis. Most likely seen in tissues with high metabolic demand: CNS. Heart. Skeletal muscle. May also be seen in other tissues e.g. pancreatic β cells. 45 4. Mitochondrial DNA mutations One disease in which mtDNA mutations have been reasonably well characterised is Parkinson’s Disease (PD). A number of deletions in mtDNA have been seen in the substantia nigra of both sporadic and early onset familial PD. Believed to be related to the high level of reactive oxygen species associated with dopamine production. These damage mtDNA leading to reduced ATP production in the dopaminergic neurones of the substantia nigra. Cancer is a multi-gene disease. There is a lot of evidence of the involvement of damaged nuclear DNA in cancer. There is evidence of point mutations in mtDNA in some cancers. The relative importance of mtDNA in cancer is unclear. 46 Summary The smallest unit of DNA is a nucleotide that consists of a nitrogenous base, pentose sugar and phosphate group. Polynucleotide chains are formed through phosphodiester bonds that link the phosphate group and sugar between nucleotides. DNA wraps around histone proteins to condense into chromatin. The DNA code undergoes transcription and translation to make functional proteins (phenotype). These are multi-step processes that are highly controlled through enzyme action. DNA is also found in the mitochondria (without introns) that largely produce proteins for respiration. Various disease states are caused by mutations to this part of the genome. 47 Questions What are the different nitrogenous bases used in nucleotide structure of DNA and RNA? What is a replication fork? Describe its structure and the directionality of DNA synthesis in it. Use a diagram to help illustrate your answer. What are the similarities and differences between nuclear and mitochondrial DNA? Outline the structure of DNA through its levels of organisation found in a cell. What bonds are found in this structure? Use a diagram to help illustrate your answer. 48 Things to consider before the next session… Re-read the lecture notes from today. Consult text books for further reading. Read the next sessions lecture notes before attending. Definitions for next time: base pairing, DNA structure, genes. Further reading: Campbell Biology. Chapter 16 and 17. 49