Transcript Slides - The University of Sydney

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
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)
Multitasking!
• Polyribosomes
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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
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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
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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
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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