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 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!

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

tRNA fmet Special tRNA and amino acid used to initiate (tRNA i or tRNA fmet ) –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)

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)

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 –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

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’)

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 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

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

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