Protein Biosynthesis • Away from the Ribosome Genetic code Charging tRNA Ribosomes • On the Ribosome Initiation complex Elongation factors Peptide bond formation Termination
Characteristics of the Code • non-overlapping • degenerate • triplet • basically universal over all living species • polar • no punctuation • subject to miscues • defines a reading frame
Garbled Garbled In frame In frame N ^ Framing the Code OLD MAN AND THE SEA Ernest Hemmingway Insert A OLD MAA NAN DTH ESE A Insert AA OLD MAAANA NDT HES EA OLD MAAANA AND THE SEA Insert AAA Insert A delete N OLD MAA NAD THE SEA
Charging tRNA Q: What is meant by Charging A: Charging means placing an amino acid on the 3’ (acceptor) end of the tRNA Q: So, what’s the big deal? A: There are 20 amino acids; the code is degenerate There could be 4 “isoaccepting tRNAs” competing for one Q: I still don’t see a problem A: One enzyme must recognize 4 different tRNA species and select the correct amino acid.
Q: One enzyme does all that? A: No, each tRNA has its own enzyme Q: What is this enzyme called? A: Its call Aminoacyl-tRNA Synthetase Q: So, there are 20 of these enzymes A: Yes Q: That makes the job a recognition a little easier then? A: Yes, but the enzymes still have to distinguish between look-alikes such as leucine and valine, glutamine and glutamate, tyrosine and phenylalanine.
Q: Are all aminoacyl-tRNA synthetases alike? A: Yes and no. Yes, they perform the same function, i.e., to recognize and transfer the correct amino acid to tRNA. Q: Why no? A: Because one class (Class I) looks for the anticodon on the tRNA, the other (Class II) looks for other features. Q: What else? A: Class I puts the amino acid on the 2’ position of the terminal ribose on tRNA, Class II only the 3’.
Q: So, how does aminoacyl-tRNA synthetase discriminate amino acids and different tRNA species? A: The key lies in the tRNA itself. Besides the anticodon, tRNAs have other bases that set them apart. These bases called “identity elements” are found in the terminal ends (acceptor stem) and internal in the tRNA. Q: Do they also proofreading? A: Yes, but sparingly Q: How sparingly? A: Enough to keep errors down to isoleucine mistaken for a valine once every 50,000 times. Ile-tRNA synthetase actually hydrolyzes the valine-AMP precursor.
L-Valine CH3 CH3 CH2-CH2 CH2-CH2 CH-COO- CH-C CH3 CH3 NH3+ NH3+ O PPi ~ O CH3 CH2 CH-COO- O-P-O-CH2 CH3 NH3+ Ad O O L-Isoleucine HO OH CH3 CH2 CH-COO- CH3CH2 NH3+ Reaction: + ATP L-Leucine Enzyme Bound tRNA
5’ 3’ 3’ 5’ 3rd position Wobble base on anticodon I C G G C A Codon-Anticodon Interactions Polarity Anticodon on tRNA Codon on mRNA 3’ 5’ Anticodon loop Alanine mRNAs are always read 5’ to 3’. mRNA 5’ 3’ (C, U)
Crevice tRNA sites Ribosomes: The Staging Areas of Protein Synthesis mRNA Ribonucleoprotein Particles 30S 70S (80S) Monosomes 50S 30S (40S) 16S RNA (18S) 23 Peptides (33) 50S (60S) 23S RNA (28S) 31 Peptides (49) 5S RNA (5S + 5.8S) * Mammalian
Polysomes Groups of ribosomes attached to a single mRNA