- Slides: 12
Translation II Lecture 14 Don’t forget the amazing role play.
Bringing in the aa-t. RNA • Uses a protein called an Elongation Factor – EF-Tu – GTP hydrolysed as t. RNA brought in and peptide bond is formed – 23 S r. RNA 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 m. RNA! • At the end, the ribosome dissociates • Note how GTP is hydrolysed at several steps – Translation is quite costly – As was transcription!
Initiation • Ribosome is separated for initial m. RNA binding – Through binding of Initiation factor (IF-3) – 3’ end of 16 S r. RNA in 30 S subunit binds m. RNA • 5’-----AGGAGGU--- • The Shine-Dalgarno sequence – Positions an AUG in the P-site – SETS THE READING FRAME • Other initiation factors – IF-1 blocks A-site and prevents t. RNA entry – IF-2 Used to bring in the first t. RNA to P-site • 30 S initiation complex formed – IFs leave once the t. RNA is in place – Allow the 50 S subunit to bind
t. RNAfmet • Special t. RNA and amino acid used to initiate (t. RNAi or t. RNAfmet) – t. RNA coupled to N-formyl-methionine – Formyl group added after met put on t. RNA – 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)
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 m. RNA from nuclease attack • stability! • Coupled transcription and translation – m. RNA 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)
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?
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 – – UUUUU makes a polypeptide containing phenylalanine AAAAA makes poly-lysine CCCCC 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
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 m. RNA is quite flexible • more so than ds. DNA where pur=pur or pyr=pyr pairs absolutely not allowed • And G can pair to U – So there are two t. RNAs for alanine • one has CGI as anti-codon, one has CGC • but note my slack order (should write 5’ to 3’)
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
More on t. RNA • t. RNA is made from DNA – There are ‘genes’ for the t. RNAs – 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
Antibiotics • Some antibiotics specifically affect procaryotic translation – – Streptomycin – binds to 30 S, prevents initiation Tetracycline – binds to 30 S, prevents t. RNA binding Chloramphenicol – inhibits peptidyl transferase of 50 S Erythromycin – binds to 50 S, prevents translocation • So they kill bugs but not eucaryotic cells
Textbook • p 171 -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. • p 176 on wobble – Including table 9 -2 • • • p 177 -8 on polycistronic m. RNA and multiple reading frames p 181 on initiation of protein synthesis p 184 on the translation of polycistronic messages p 185 on polysomes p 186 on coupled transcription-translation – we will do the eucaryotic cap stuff next lecture • p 188 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