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BIOL2202 – Module 1 Summary Translation 1. The small & large ribosome subunits assemble around the mRNA 2. The mRNA passes through the ribosome, positioned by the small subunit, so it can be read in codons 3. tRNA enters the ribosome at the A site and is tested for a codon/anticodon match 4. If there’s a correct match, the tRNA shifts to P site and the amino acid is attached to the protein chain 5. Spent tRNA is moved to the E site and is ejected to be recycled 6. As the protein grows, it folds into a precise shape thanks to IMF’s, though it initially exits the ribosome like a noodle tRNA Properties  Cloverleaf secondary structure  Amino acid covalently attached to the 3’ end of the tRNA  Each amino acid has at least one tRNA gene that encodes tRNAs used specifically for that amino acid  Unique in that they are written 3’ to 5’, because they need to be complimentary to mRNA  Stop codons do not match with a tRNA, but a protein called a release factor Abnormal Bases in tRNA’s  These are created by heavy post-transcriptional modification of tRNA  Modified bases allow tRNA greater flexibility of function than possible with only A, U, G and C  ~80 are known  Best-known is inosine (I), created by deamination (removal of amino group) of adenine into hypoxanthine – forms bonds with A, U, and C, but not G  Assists in tertiary structure formation In-Vitro Protein Synthesis (Determining the Genetic Code)  Grind E. coli cells with powdered alumina  Centrifuge to pellet (separate) the cell wall  Collect cell lysate  Treat with DNase to stop mRNA synthesis  Existing cellular mRNA will degrade since it is highly unstable  Add the synthetic RNA template polyuracil, which reads UUUUU..., and one amino acid labelled with the radioactive carbon atom C14  Which combinations insert the C14 into the protein?  For hetero polymers, simply repeat until you’re sure only two amino acids can be produced  For codes reading GUGGUGGUG and similar, three are three possible codons – repeat above, but check for three amino acids  Combine the data to find specific combinations (a logic game)

Wobble  The E. Coli genome only has 40 tRNA genes, yet there are 61 codons in the standard genetic code which specify amino acids – this must mean that some tRNAs are able to recognise more than one codon!  The Wobble Hypothesis was proposed by Francis Crick in 1966 to explain how one tRNA molecule could recognise more than one codon – the last base in an anticodon (the sequence on the tRNA molecule), i.e. position 1, can “wobble”, meaning there are extra base-pair options compared to the standard A=U and G=C tRNA base 1st position in anticodon C A U G I 

mRNA base 3rd position in codon G U A or G U or C U, C or A

The use of wobble is limited by the fact that tRNA’s must only recognise codons for a single amino acid, or the genetic code would be compromised

Naming DNA Strands  5’ -> 3’ o Coding o Forward o Sense o Crick o Non-template  3’ -> 5’ o Non-coding o Reverse o Antisense o Watson o Template Reading Frames

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mRNA has 3 reading frames, while double-stranded DNA has 6

Key Phrases Start of an open reading frame/start of a protein/N-terminus of a protein End of an open reading frame/the end of a protein/the C-terminus of a protein Middle of an open reading frame/middle of a protein Candida albicans/Prokaryote (or wellknown species such as E. coli) Disulphide bond A serine-rich protein (or some other amino acid) A lysine residue (or some other amino acid)

What you might need to look for A start codon but no stop codon A stop codon

No stop codon Alternative codon usage A cysteine residue Serine residues (or the amino acid mentioned) A lysine residue (or the amino acid mentioned)

Mutation  Mutation is made inevitable by the spontaneous deamination of cytosine (conversion to uracil) and the spontaneous hydrolysis of purines (guanine and adenine)  Spontaneous mutations occur due to inherent metabolic errors or unknown agents in the environment – they “just pop up on their own”   

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Induced mutations result from exposure to mutagens Germinal mutations occur in germ-line cells and will be transmitted through the gametes to the progeny Somatic mutations occur in somatic cells; the mutant phenotype will occur only in that cells and its direct descendants, and is not transmitted to the progeny of the organism Missense mutations are caused by an amino acid substitution which leaves the protein defective Nonsense mutations cause a stop codon, which prematurely ends the protein and often leads to a loss of funciton Experiment below demonstrates that mutations arise before selection, not after

Stationary Phase Mutagenesis in Bacteria and Fungi    

This is a process that increases the rate of spontaneous mutation in a cell, potentially creating a mutant with a selective advantage in a certain environment Occurs when populations of bacteria or fungi stop growing This is the ability to increase mutagenesis in order to overcome environmental stress in the face of population death (the entire genome gets mutagenized! Driven by DNA Polymerase V – see The SOS response in E. Coli

Reversing Mutation  Forward mutation: mutation of a wild-type allele to a mutant allele  Reverse mutation (reversion): a second mutation that restores the original phenotype o Back mutation: a second mutation at the same site o Suppressor mutation: a second mutation at a different location in the genome Types of Alleles  Isoalleles have negligible or no effect on phenotype  Null alleles result in no gene product or totally non-functional gene products  Neutral alleles have no effect on the fitness of the organism  Recessive lethal mutations are lethal in the homozygous recessive state Multigenic Control of Phenotypes  Some enzymes may be multimeric (several structures held together by weak bonds); multiple genes could control the enzyme via a combination of subunits  Other genes may be involved in the regulation of a gene product  Phenotypes are usually multigenic Properties of Amino Acids  There are 20 amino acids split into five categories based on their R group 

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Synonymous mutation: new codon, but no amino acid change (caused by the degeneracy of the genetic code) Non-synonymous mutation, conservative substitution: a new codon causing a new amino acid which has similar physical properties to the one it replaced Non-synonymous mutation, non-conservative substitution: a new codon causing a new amino acid which does not have similar physical properties to the one it replaced

Tautomeric Shifts  Tautomeric shifts can cause substitution mutations because of a change from “keto form” to “enol form”

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Transition mutations vs transversion mutations:

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Transition mutations occur far more often, because they are caused by tautomeric shifts 5-Bromouracil is a base analogue which can behave like cytosine in enol form or thymine in keto form – it can casually swap isomers, meaning that if an organism is exposed, its DNA can start to change across replications

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Mutagenesis via Ultraviolet Radiation

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The hydrolysis of cytosine (producing cytosine hydrate) may cause mispairing during replication The cross-linking of adjacent thymine bases via UV radiation can form thymine dimers, which block DNA replication and activate an error-prone DNA repair mechanism

Suppressor Mutations  This is when a second mutation masks the deleterious effects of a first mutation  A second mutation within the same gene but a different codon is an intragenic suppressor mutation  A second mutation within a different gene is an intergenic suppressor mutation Detecting Mutagens - The Ames Test  The test done with histidine auxotrophic mutants of Salmonella – mutagens will cause reversions, producing histidine prototrophs  To conduct the test, homogenise a rat liver and combine with bacteria, then add the mixture to a plate with a medium lacking histidine and incubate  Repeat the above steps, but add test chemical to the disk  Substitution mutagens will typically produce no effect, while frameshift mutagens will cause a reversion, producing histidine prototrophs and preventing colony growth...