How is the code contained in mRNA translated into a protein?
Structure and function of transfer RNA's
- tRNA's have two functions:
- To chemically link to a particular amino acid (covalent)
- To recognize a specific codon in mRNA (non-covalent) so that its attached amino acid can be added to a growing peptide chain
Amino-acyl tRNA synthetases
- Function is to "charge" tRNA molecules; i.e. to chemically link a specific amino acid to its associated tRNA molecule.
- There is one amino-acyl tRNA synthetase per amino acid (they are quite specific).
- There is potentially more than one tRNA per amino acid.
- There is potentially more than one codon per tRNA.
Structure of tRNA's
- 70-80 nucleotides long
- Form a series of stem/loop secondary structures
- tRNA's are synthesized with the standard bases AGCU. However, after synthesis several bases may be modified:
- Uridylate may be methylated to produce Thymidylate
- Uridylate may be rearranged to produce pseudouridylate (i.e. ribose attached to Carbon 5 instead of Nitrogen 1).
- Guanidylate may be methylated at different positions.
- The amino acid is attached at the 3' end of the tRNA to either the 2' hydroxyl or the 3' hydroxyl.
- Class I amino-acyl tRNA synthetases attach their associated amino acids to the tRNA 2' hydroxyl (NOTE: typically the hydrophobic amino acids)
- Class II amino-acyl tRNA synthetases attach their associated amino acids to the tRNA 3' hydroxyl (NOTE: typically hydrophilic amino acids)
Figure 2.8.1: tRNA
- If perfect Watson-Crick base pairing were required at the codon/anti-codon triplet then 61 different tRNA's would be required.
- We know this is not the case, therefore a single tRNA anti-codon must be able to recognized several different mRNA codon triplets.
- This greater recognition of tRNA is possible due to "wobble" basepair interactions at the third base in the codon/first base in the anti-codon:
Figure 2.8.2: Codon wobble
Possible "wobble" codon base pairing (in addition to Watson-Crick):
- U - G
- I - C
- I - A
- I - U
- Where U, G, A and C can be in either the codon (mRNA) or anti-codon (tRNA)
- I (inosine) can be found in the anti-codon.
Recognition of amino acids by amino-acyl tRNA synthetases
- Appears to involve not only the anti-codon triplet but significant other contacts as well (mostly involving the acceptor stem region).
- The mRNA with its encoded information and the individual tRNAs loaded with their amino acids are brought together by a mutual affinity for an RNA-protein complex called the Ribosome.
- The rate of protein synthesis by a ribosome is approximately 3-5 amino acids/minute.
- For example, a large protein (e.g. Titin, 30,000 amino acids) takes 2-3 hours to make.
- Ribosomes are composed of individual ribosomal RNA (rRNA) molecules and more than 50 accessory proteins, with a general prokaryotic organization of a small subunit (30S) and a large subunit (50S).
Translation of mRNA to proteins
- Protein synthesis is usually considered in three steps:
AUG is the initiation signal in mRNA
- The first event of the initiation stage is the attachment of a free molecule of methionine (Met) to the end of a tRNAMet by a specific aminoacyl-tRNA synthetase.
- There are at least two types of tRNAMet:
- tRNA i Met: can initiate protein synthesis (at AUG met codon)
- tRNA Met: can incorporate Met residues during on-going protein synthesis (at AUG met codon)
- Methionine tRNA synthetase attaches Methionine to both tRNA molecules.
- Only methionyl-tRNA i Met can bind to the small ribosomal subunit to begin the process of protein synthesis.
- In bacteria, the amino group of the methionine in methionyltRNAiMet is formylated.
- The Met-tRNA i Met, along with a protein-GTP complex and the small (30S) ribosomal subunit bind to the mRNA at a specific site, near the AUG initiation codon.
Initiation of protein synthesis
- In most prokaryotes an RNA component (16S rRNA) in the small rRNA subunit (30S) recognizes and hybridizes to a specific sequence on the mRNA called the Shine-Dalgarno sequence:
- The Shine-Dalgarno sequence is thus a ribosome binding site which is necessary for the intiation of translation.
- Note that the ribosome does not bind at the AUG start codon, but 5-10 nucleotides upstream.
- The Shine-Dalgarno sequence can be located anywhere within an mRNA.
- A series of initiation factors, Met-tRNAiMet , mRNA and the 30S (i.e. 16S component) ribosomal subunit are necessary for formation of the 30S initiation complex.
- The large (50S rRNA) rRNA binds along with release of initiation factors 1 and 2, and hydrolysis of GTP, to form the 70S inititation complex:
Figure 2.8.3: Initiation complexes
- In the first part of the elongation step of translation, the ribosome moves along the mRNA to position the fMet residue to the P site (peptidyl site) in the 50S subunit.
- This allows the second codon of the mRNA to be positioned in the A site (amino acyl tRNA site).
- The appropriate charged tRNA (with amino acid) specified by the second codon is positioned in the A site of the 50S subunit.
- Next peptide bond formation is synthesized and the tRNA in the A site (which is covalently attached to the nascent polypeptide) is translocated to the P site.
- This process requires GTP and the G elongation factor protein (prokaryotes).
- The process is repeated.
- When a stop codon is reached the polypeptide is hydrolyzed away from the last tRNA.
- The peptide is released and the ribosome typically dissociates.
- This process requires GTP and three different termination factors (TF's; only one required in Eukaryotes)