2.8: mRNA Translation

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Mar 6, 2021
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- 2.7: Transcriptional Regulation: Transcription termination, the trp operon
- 3. Biotechnology 1

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How is the code contained in mRNA translated into a protein?

Structure and function of transfer RNA's

tRNA's have two functions:
1. To chemically link to a particular amino acid (covalent)
2. 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.

Amino Acids	Amino-acyl tRNA synthetases	tRNA's	Codons
20	20	30-40 (prokaryotes) 50 (eukaryotes)	61 (3 stop codons)

Conclusions:

There is one amino-acyl tRNA synthetase per amino acid (they are quite specific).
There is potentially more than one tRNA per amino acid.

Therefore, amino-acyl tRNA synthetases must be able to recognize more than one tRNA.

There is potentially more than one codon per tRNA.

Therefore each tRNA must be able to recognize more than one codon (there is not a unique tRNA for each codon).

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:
1. Uridylate may be methylated to produce Thymidylate
2. Uridylate may be rearranged to produce pseudouridylate (i.e. ribose attached to Carbon 5 instead of Nitrogen 1).
3. 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.
1. Class I amino-acyl tRNA synthetases attach their associated amino acids to the tRNA 2' hydroxyl (NOTE: typically the hydrophobic amino acids)
2. Class II amino-acyl tRNA synthetases attach their associated amino acids to the tRNA 3' hydroxyl (NOTE: typically hydrophilic amino acids)

Screenshot (273).png

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:

Screenshot (274).png

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.

For example, the codons UUU and UUC are both recognized by the tRNA which has GAA in the anti-codon position (making either G - C, or G - U base pairings).

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).

Ribosomes

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:

Initiation
Elongation
Termination

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 tRNA^Met by a specific aminoacyl-tRNA synthetase.
There are at least two types of tRNA^Met:
1. tRNA _i^Met: can initiate protein synthesis (at AUG met codon)
2. 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 methionyltRNA_i^Met 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:

mRNA 5' -UAAGGAGG -(5-10 nucleotides)-AUG 3' 16S rRNA OH-AUUCCUCC -(~1400 nucleotides)-5'

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-tRNA_i^Met , 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:

Screenshot (275).png

Figure 2.8.3: Initiation complexes

Elongation

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.

Termination

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)