DNA & RNA

LECTURE 13 DNA   Describe evidence that DNA carries genetic information Frederick Griffith (1928) performed studies using two strains of the Streptococcus pneumoniae bacterium - one pathogenic, the other non-pathogenic. Pathogenic bacteria injected into a mouse caused the mouse to die, while non-pathogenic bacteria and heat-killed pathogenic bacteria did not affect the mouse. However, when Griffith mixed together heat-killed pathogenic cells and non-pathogenic cells, the mouse died, and living pathogenic cells could be found in the mouse's blood. This suggested that the living non-pathogenic cells were inheriting nformation from the dead pathogenic cells. Later work by other scientists identified the transforming substance as DNA. Hershey and Chase's studies (1952) showed further evidence that DNA carries genetic information with their studies of viruses and E. coli. Hershey and Chase incorporated radioactive sulfur atoms into the protein of the bacteriophage and radioactive phosphorus into its DNA to see if it is proteins or DNA that enters the E. coli. They found that DNA entered the cell while proteins did not, and after some time the E. coli released new phages that contained radioactive phosphorus. Hershey and Chase concluded that it was DNA that was carrying genetic information to allow the cells to produce new viral DNA and proteins. In 1950, Erwin Chargaff discovered that the base composition of DNA varies between different species, further suggesting that DNA is the genetic material.   Show that DNA is an anti-parallel double-stranded polynucleotide in which base-pairing plays a crucial role DNA consists of two anti-parallel (run in opposite directions, 5'-3' and 3'-4') strands made up of a sugar-phosphate backbone and nitrogenous bases joining the two strands, twisted into a double helix. One sugar-phosphate molecule joined to a nitrogenous base makes up one nucleotide. The sugar-phosphate molecules are joined to each other by covalent bonds (phosphodiester bonds), and the base pairs are joined to each other by hydrogen bonds. Bases A and G are purines (double-ringed), and bases C and T are pyrimidines (single-ringed). A bonds with T (2 H-bonds) and G bonds with C (3 H-bonds). Evidence can be seen in that there are equal ratios of A and T bases and G and C bases in DNA. https://www.youtube.com/watch?v=xozXUIVeSpY&list=PLU9wEyzVsaZXPYJKqKM7qyb4D-vosDwAn   Explain the Meselson-Stahl experiment to demonstrate semi-conservative replication of DNA Meselson and Stahl cultured E. coli over several generations in a culture containing nitrogen isotope 15N. They then transferred the bacteria to a culture containing only 14N, which is lighter than 15N. They extracted DNA from samples taken after the first and second DNA replication and centrifuged the DNA samples to separate the DNA by density. The sample taken after the first replication showed one band of hybrid 15N and 14N DNA, while the sample taken after the second replication showed both light DNA and hybrid DNA. This concluded that DNA replication is semi-conservative.   Discover that DNA polymerase and other proteins achieve DNA replication DNA replication: https://www.youtube.com/watch?v=NX9kwdQ3R6s; https://www.youtube.com/watch?v=UizoPujYRuU Topoisomerase: breaks, swivels, and rejoins parental DNA ahead of replication fork, relieving strain caused by unwinding. Helicase: unwinds DNA strands. Single-strand binding protein: binds to and stabilises a single-stranded DNA until it is used as a template. Primase: synthesises RNA primers on the lagging strand. DNA polymerase III: synthesises new DNA strand by adding nucleotides in a 5' to 3' direction. DNA polymerase I: replaces RNA primers with DNA on lagging strand. DNA ligase: forms bond between DNA fragments   Explain that DNA replication is a very accurate process but that rare mistakes can lead to mutations DNA polymerase proofreads nucleotides added to the template strand during the process of building the new strand. If the polymerase detects a mismatched nucleotide, it removes the nucleotide before continuing synthesis. In the case that the mismatched nucleotide evades proofreading by polymerase, it can be removed by other enzymes that replace incorrectly matched nucleotides. DNA nucleases cut out (excise) mismatched base pairs and the resulting gap is filled in with nucleotides by DNA polymerase and ligase. DNA molecules can be altered after replication by harmful chemical and physical agents (eg X-rays) and can change under normal cellular conditions, and thus constant care is required. 

LECTURE 14 RNA Describe the evidence for the link between genes and proteins as shown by studies of mutants. Garrod (1902) suggested that inherited diseases are a result of the body's inability to synthesise a specific enzyme. Beadle and Tatum (1920s) expanded on this hypothesis through studies on Neurospora crassa, a bread mold. They exposed wild-type bread mold to X-rays and observing how mutated mold cells could survive in minimal medium compared to wild-type mold. While wild-type mold was able to survive in minimal medium (they could produce all necessary amino acids from the inorganic salts, glucose, and vitamins included in the medium), mutants needed a complete growth medium containing all amino acids in order to survive. Further studies allowed Beadle and Tatum to determine which amino acid the cells were unable to synthesise based on mediums lacking specific amino acids. Through these studies they were able to deduce that mutations had impacts on the synthesis of enzymes required for the synthesis of specific amino acids.   Describe the copying action of RNA polymerases and explain the role of the DNA template during transcription. RNA polymerases work similarly to the DNA polymerases of DNA replication. They attach to the DNA strands and unwind them, while adding nucleotides to the growing RNA molecule in a 5'-3' direction based on the template DNA strand. The RNA molecule is made up of complementary bases to the DNA template strand. In prokaryotes, there is only one RNA polymerase, in eukaryotes there are more. The transcription of DNA to make mRNA is carried out by RNA polymerase II. Transcription occurs in three steps: initiation, elongation, and termination.   Discover how RNA polymerases find the start of a gene and make the transcript of the template strand of DNA in the correct direction. Unlike DNA replication, primers are not needed for polymerase to find the starting point of transcription. Instead, the starting point is indicated by promoter sequences. In bacteria, the sequence that signals the end of transcription is called the terminator. The terminator is located downstream from the promoter (5' --> 3'). In eukaryotes, the terminator is a polyadenylation signal which is added to the pre-mRNA strand. The RNA polymerase binds to the promoter sequence in a precise location and orientation, determining where transcription starts and which strand will be the template strand. In eukaryotes, transcription is mediated by transcription factors; RNA polymerase does not bind to a promoter until transcription factors are attached to the promoter. The whole complex is called the transcription initiation complex. A eukaryotic promoter commonly includes a TATA box about 25 nucleotides from the transcriptional start point.   Describe how RNA transcripts in eukaryotic cells are processed into their mature functional forms and transported to their correct location in the cell. In eukaryotes, before translation, pre-mRNA must be processed into mature mRNA before it leaves the nucleus to be translated. This processing involves adding a guanine cap to the 5' end of the RNA molecule, and a poly-A tail (made up of adenine nucleotides) to the 3' end of the molecule. The cap and poly-A tail facilitate the transport of the mRNA from the nucleus to the cytoplasm, protect the mRNA from degradation by hydrolytic enzymes, and help ribosomes attach to the 5' end of the mRNA molecule. The processing of pre-mRNA also involves RNA splicing, in which introns are cut out from the RNA by a spliceosome and the exons are bound together.   Describe a mature mRNA transcript showing the relative locations of 5'- and 3'- ends, start and stop signals (codons). A mature mRNA molecule will have, on average, 1200 nucleotides that will be translated into a polypeptide. mRNA has a 5' cap, made of a modified form of guanine, and a poly-A tail made up of 50-250 adenine nucleotides. There will be UTRs (untranslated regions) at both the 5' and 3' ends of the exon in the middle of the mRNA molecule, before and after the start and stop codons. The start codon is AUG, which gets translated to methionine (which may be removed from the polypeptide by enzymes later), while the stop codons are UAA, UGA, and UAG.  

LECTURE 15 PROTEIN SYNTHESIS Explain that cells need to make polypeptides of defined length and sequence (from previous block). Polypeptides need to be a specific length and sequence in order to make the proteins the organism needs to carry out its functions.   Describe the importance of base-pairing in ensuring the accuracy of protein synthesis. Complementary base-pairing is important so that the correct amino acids could be added to the polypeptide chain in the correct sequence. For this reason, tRNA molecules contain a specific anti-codon for the codons of mRNA molecules that correspond to the amino acid that this tRNA molecule brings to the ribosome. Aminoacyl-tRNA synthetases ensure that the correct amino acid is attached to the tRNA molecules.   Describe the machinery (ribosome), raw materials (aminoacyl-tRNAs) and instruction tape (mRNA) for protein synthesis (translation). Ribosome: Consists mostly of rRNA (ribosomal RNA) and proteins. Is made up of two subunits - a large subunit and a small subunit, that only come together once an mRNA binds to the small subunit. When this happens, initiation factors and the hydrolysis of GTP (for energy) bring the large subunit and small subunit together. Together they form a translation initiation complex. Ribosomes have three sites: E, P, and A. The P site holds a tRNA molecule that brings in the first amino acid (Met) (part of the translation initiation complex), and holds the growing polypeptide chain, while other tRNA molecules enter the A site and attach their amino acid to the chain. tRNA exits through the E (exit) site. Aminoacyl-tRNA: A single stranded RNA molecule bent into an L-like 3D shape (using H-bonds), with an amino acid attachment site at one end and an anti-codon at the other. mRNA: Contains start and stop codons as well as the codons translated to a polypeptide chain.    Explain the main characteristics of the genetic code which defines the association of codons with anti-codons. Codons consist of 3 nitrogenous bases that each have a corresponding complementary base, which form an anti-codon. Each codon corresponds to one amino acid. Codons are almost universal - with some exceptions, most codons code for the same amino acids in all organisms.   Explain that, for proper cell function, proteins must be processed and targeted to their correct cellular locations. As the polypeptide is being synthesised, it starts to coil into a 3-dimensional shape (secondary and tertiary structure) as a result of interactions of its amino acid sequence. Chaperonin (chaperone protein) helps the polypeptide fold correctly. Other post-translational modifications may be carried out: removal of amino acids from the end of the chain, chemical modification through the attachment of sugars, lipids, phosphate groups, etc., enzymatic cleaving of the polypeptide chain into two or more chains, different polypeptides may be joined together (quarternary structure). Free ribosomes make proteins that stay in the cytosol, bound ribosomes (bound to rER) make proteins that are used in the endomembrane system or transported out of the cell. All translation begins as translation on free ribosomes, but polypeptides of proteins that are destined for the endomembrane system or secretion are marked by a signal peptide which targets the protein to the ER. The signal peptide is recognised by a protein-RNA complex called SRP (signal-recognition particle) that bring the ribosome to a receptor protein built into the ER membrane and is part of a multiprotein translocation complex. The growing polypeptide snakes across the membrane into the ER lumen via a protein pore, and the signal peptide is removed by an enzyme.If the polypeptide is to be secreted, it is released into solution within the ER lumen, if it is to be a membrane protein, it remains partially embedded in the ER membrane. In both cases, it travels in a transport esicle to its destination.