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Translation II

Translation II. Lecture 14. Don’t forget the amazing role play. Bringing in the aa-tRNA. Uses a protein called an Elongation Factor EF-Tu GTP hydrolysed as tRNA brought in and peptide bond is formed 23S rRNA actually catalyses the peptide bond formation

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Translation II

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  1. Translation II Lecture 14 Don’t forget the amazing role play.

  2. Bringing in the aa-tRNA • Uses a protein called an Elongation Factor • EF-Tu • GTP hydrolysed as tRNA brought in and peptide bond is formed • 23S rRNA actually catalyses the peptide bond formation • EF-G catalyses movement of the ribosome • Again using GTP  GDP • Probably better to say that it moves the mRNA! • At the end, the ribosome dissociates • Note how GTP is hydrolysed at several steps • Translation is quite costly • As was transcription!

  3. Initiation • Ribosome is separated for initial mRNA binding • Through binding of Initiation factor (IF-3) • 3’ end of 16S rRNA in 30S subunit binds mRNA • 5’-----AGGAGGU---- • The Shine-Dalgarno sequence • Positions an AUG in the the P-site • SETS THE READING FRAME • Other initiation factors • IF-1 blocks A-site and prevents tRNA entry • IF-2 Used to bring in the first tRNA to P-site • 30S initiation complex formed • IFs leave once the tRNA is in place • Allow the 50 S subunit to bind

  4. tRNAfmet • Special tRNA and amino acid used to initiate (tRNAi or tRNAfmet) • tRNA coupled to N-formyl-methionine • Formyl group added after met put on tRNA • Formyl group forms a sort of mini-peptide bond at the N-end • New proteins in bugs have N-formyl-met at the end • Sometimes this is hydrolysed off (50% of the time)

  5. Multitasking! • Polyribosomes • Always several translating at once • Once the first 25 amino acids cleared • So one ribosome every 80 nucleotides • See pictures in book • Ribosomes may protect mRNA from nuclease attack • stability! • Coupled transcription and translation • mRNA made 5’ 3’ • Translated in same direction • So can be translated as it is transcribed • Speed of both is 45 nucleotides per second • Doesn’t happen in eucaryotes (where there is a nucleus)

  6. Reading Frames • Some viruses can have multiple reading frames • Reading frame set by AUG used to initiate • Enables many proteins to be made from one transcript • very efficient use of DNA! • But imagine the effect of a mutation! • How seriously does it constrain the amino acid sequence in each protein?

  7. The Genetic Code • How do we know that a triplet code is used? • Code worked out by synthesising RNAs and seeing what peptides they made • Incubation of cell extracts with the RNAs and mixtures of amino acids • UUUUUUUUUU makes a polypeptide containing phenylalanine • AAAAAAAAAA makes poly-lysine • CCCCCCCCC makes poly-proline • Later triplet RNAs were made and tested • There are twenty amino acids but 64 codons • What happens to the unused 44 codes? • See Table 9.1 in textbook • CCA, CCC, CCG, CCT all code proline • GCA, GCC, GCG, GCT all alanine

  8. The Spare Codons • The code is DEGENERATE or REDUNDANT • A rather negative way of saying that there are synonyms! • The redundancy is normally in the last base • First two bases in codon well paired • This is called WOBBLE • Due to the presence of INOSINE • Which can pair to A, U or C • And because mRNA is quite flexible • more so than dsDNA where pur=pur or pyr=pyr pairs absolutely not allowed • And G can pair to U • So there are two tRNAs for alanine • one has CGI as anti-codon, one has CGC • but note my slack order (should write 5’ to 3’)

  9. The Code is Universal • Only two amino acids have one codon • Met and Trp • Actually prevented from wobble by modification of bases • So mutations in DNA often don’t affect the amino acid sequence • Especially if in the last nucleotide in the codon • But it’s impossible to deduce the nucleic acid sequence from a protein sequence! • Pretty much all life forms use the same code • eg, GCC always encodes alanine • But slight variations in mitochondria • So human genes can be read in bacteria and pig genes can be read in plants • If this wasn’t the case, Biotechnology would be much more difficult

  10. More on tRNA • tRNA is made from DNA • There are ‘genes’ for the tRNAs • Long RNA transcribed • Not translated but cleaved by RNases • Which, themselves, are made up of RNA • Note how many fundamental processes are catalysed by RNA

  11. Antibiotics • Some antibiotics specifically affect procaryotic translation • Streptomycin – binds to 30S, prevents initiation • Tetracycline – binds to 30S, prevents tRNA binding • Chloramphenicol – inhibits peptidyl transferase of 50S • Erythromycin – binds to 50S, prevents translocation • So they kill bugs but not eucaryotic cells

  12. Textbook • p171-2 on the Genetic Code • You don’t need to know all the codes in Table 9-1, but you should know how to read such a table and you should reflect on the degeneracy. • p176 on wobble • Including table 9-2 • p177-8 on polycistronic mRNA and multiple reading frames • p181 on initiation of protein synthesis • p184 on the translation of polycistronic messages • p185 on polysomes • p186 on coupled transcription-translation • we will do the eucaryotic cap stuff next lecture • p188 on antibiotics • it’s not necessary to know what each antibiotic does, just that many antibiotics can interfere with various parts of the translation process • a good exam question would be to get you to give you a scenario and get you to predict which step the antibiotic was affecting • or to tell you what step an antibiotic affected and get you to predict the results

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