Translation Lecture 13

Converting mRNA to Protein DNA Information in sequence of bases Protein Sequence of amino acids mRNA How do we convert the sequence of bases in mRNA to a sequence of amino acids in protein?

The Message on mRNA A group of three bases codes for a specific amino acid –CODON –eg, UUU for phenylalanine, CCC for proline, GGG for glycine Sixty four different codons –Includes START and STOP codons AUG - start UAG, UAA, UGA - stop –Other 60 easily enough for 20 amino acids –So what happens to the extra codons? THE CODON-AMINO ACID PAIRS ARE CALLED THE GENETIC CODE –Code is almost universal Slight variations in mitochondria Biotechnology would be impossible if the code varied between species! –We’ll look at it in more detail later

Adapter Molecule Something that can translate a small sequence of nucleotides to an amino acid Transfer RNA –70-90 nucleotides –Heavily folded and intra-molecular base pairs Lots of loops Often drawn as a ‘clover leaf’ (eg, fig 9-4) Sequence to recognise bases on mRNA –ANTI-CODON Needs to have amino acid attached –At the 3’ end –Amino acid attached to 3’OH –Synthesis is a two step process 5’ 3’ O Anti-codon -C-CH-NH 2 = O R Aminoacyl tRNA

5’ end 3’ end anti-codon arms anti-codon D arm acceptor stem variable loop T-psi-C arm

Activation of Amino Acid A P OH H 2 N-CH-C-O- = O R Aminoacyl AMP A PPP OH ATP H 2 N-CH-C-OH = O R Amino acid PP pyrophosphate 2P

Making the aminoacyl-tRNA 5’ 3’ OH Anti-codon A P OH H 2 N-CH-C-O- = O R Aminoacyl AMP 5’ 3’ O-C-CH-NH 2 = O R A P OH AMP Aminoacyl-tRNA tRNA Draw thus.. 3’ N

Aminoacyl tRNA synthesis Need to make sure that the CORRECT amino acid is attached to a tRNA –Incorporation of amino acid is purely based on codon:anti-codon pairing –So if the wrong amino acid is on the tRNA then the wrong amino acid will be put into the protein Aminoacyl-tRNA synthetases –Specific enzyme for each tRNA/amino acid pair –Have proof reading ability Note the isoleucine/valine example in the textbook –Recognise the correct tRNA through nucleotides in the side loops of tRNA (recognition regions) Coupling of the amino acid to tRNA involves an input of energy

Using the tRNAs The aminoacyl tRNAs attach to the mRNA –With codon:anti-codon pairing –Then the amino acids react to form peptide bonds Occurs in a stepwise manner –One tRNA at a time rather than all the tRNAs lining up! –All catalysed by a large assembly of RNA and protein called the RIBOSOME The ribosome moves down the mRNA –5’  3’ direction –Bringing in one tRNA at a time, forming peptide bonds as it goes –Protein gets longer as the ribosome moves down the mRNA

The Machinery The ribosome Large assembly of RNA and protein that binds the mRNA and brings in the amino acids –The RNA is called rRNA Very large – measured in Svedbergs 30S –21 proteins and 16S RNA 50S –34 proteins and 23S & 5S RNA Will spontaneously self assemble from constituent parts –Proteins form the scaffold, RNAs do the work

N N N N N aa-tRNA sitting in P-site aa-tRNA coming into A-site aa-tRNAs sitting next to each other on mRNA peptide bond forms dipeptide on tRNA in A-site dipeptide now on tRNA in P-site new aa-tRNA will come in to A-site cycle repeats A A P P PA AP in fact, the ribosome temporarily holds the empty tRNA in an E- site before discharge ribosome moves along – kicking out the blank tRNA

Summary With an aminoacyl-tRNA at the P-site In comes a new aa-tRNA to the A-site A peptide bond forms –Between the NH2 of the amino acid at the A-site and the carbonyl carbon of the amino acid at the P-site The ribosome moves along the mRNA –Or at least they move relative to each other! The tRNA that was in the A-site is now in the P-site –And this tRNA is carrying the peptide And the old, empty tRNA that used to be in the P-site leaves –There is a specific exit site for this And so a new tRNA comes into the vacant A-site Process continues until a stop codon is reached –no tRNA binds here –Instead, a release factor (RF) binds and hydrolyses the polypeptide from the tRNA in the P-site

Text Book All of p – Introduction and Outline –but don’t worry about the eucaryotic stuff just yet p171 is more related to the next lecture but you might as well read it now –NOTE THE MISTAKE in Fig 9.2 – the CODING STRAND is not the directly transcribed strand!! p on tRNA and aminoacyl synthetases p175 is really interesting about –which bits of tRNA are involved in recognising codons –how proof reading occurs p on Ribosomes –but don’t worry about eucaryotic ribosomes just yet p181 – the process of protein synthesis –starts with initiation which we will do in the next lecture –So leap straight to p and Figure 9-13 –Stopping (with Stop codons!) at the top of page 184

Advanced Only Nirenberg and Matthaei –Early 1960s Incubation of cell free extracts –All 20 amino acids –One radioactively labeled amino acid –Synthetic polynucleotide Made with polynucleotide phosphorylase No template Random order based on substrate ratio –Precipitate proteins made AAA  lysine, UUU  phe, CCC  pro –Why didn’t GGG work?

To each tube, add all amino acids aspgluhislysprothrphe 等等 Also, one radioactive amino acid to each tube Add synthetic polynucleotide. Incubate. Precipitate the protein Assess how much of the radioactive amino acid has been incoroporated

More Complex Polymers Mixture of 5 parts A to 1 part C Random length –AAA, ACA, AAC, CAA, etc –With high A containing triplets more likely Compare actual degree of incorporation with predicted frequency of triplets –Expressed relative to AAA/lysine

Results Amino acidObservedSuggestedExpected Asp24A2CA2C20 Glu24A2CA2C20 His6AC 2 4 Pro7CCC*, AC 2 5 Thr26AC 2, CA 2 24 Lys100A3A3 * CCC was already known to be Pro from experiments with poly-C

Later Expts Methods for making polynucleotides with repeating sequences –CACACACACACACA –Read as mixture of thr and his –So if AC 2 is his, then CAC must be his And ACA must be thr Binding of aminoacyl-tRNAs to specific trinucleotides –Ribosome, radioactive aa-tRNA and trinucleotides