Gene Translation: RNA -> Protein

The problem: How does a particular sequence of nucleotides specify a particular sequence of amino acids?

The answer: by means of transfer RNA molecules, each specific for one amino acid and for a particular triplet of nucleotides in mRNA called a codon. The family of tRNA molecules enables the codons in a mRNA molecule to be translated into the sequence of amino acids in the protein.

This image shows the structure of alanine transfer RNA (tRNA^ala) from yeast. It consists of a single strand of 77 ribonucleotides. The chain is folded on itself, and many of the bases pair with each other forming four helical regions. Loops are formed in the unpaired regions of the chain. (The bases circled in blue have been chemically-modified following synthesis of the molecule.)

At least one kind of tRNA is present for each of the 20 amino acids used in protein synthesis. (Some amino acids employ the services of two or three different tRNAs, so most cells contain as many as 32 different kinds of tRNA.) The amino acid is attached to the appropriate tRNA by an activating enzyme (one of 20 aminoacyl-tRNA synthetases) specific for that amino acid as well as for the tRNA assigned to it.

      Anticodon:  3' CGA  5'
      Codon:      5' GCU  3'

The RNA Codons

Note:

• Most of the amino acids are encoded by synonymous codons that differ in the third position of the codon.
• In some cases, a single tRNA can recognize two or more of these synonymous codons.
• Example: phenylalanine tRNA with the anticodon 3' AAG 5' recognizes not only UUC but also UUU.
• The violation of the usual rules of base pairing at the third nucleotide of a codon is called "wobble"
• The codon AUG serves two related functions
  • It begins every message; that is, it signals the start of translation placing the amino acid methionine at the amino terminal of the polypeptide to be synthesized.
  • When it occurs within a message, it guides the incorporation of methionine.
• Three codons, UAA, UAG, and UGA, act as signals to terminate translation. They are called STOP codons.

The Steps of Translation

1. Initiation

• The small subunit of the ribosome binds to a site "upstream" (on the 5' side) of the start of the message.
• It proceeds downstream (5' -> 3') until it encounters the start codon AUG. (The region between the cap and the AUG is known as the 5'-untranslated region [5'-UTR].)
• Here it is joined by the large subunit and a special initiator tRNA.
• The initiator tRNA binds to the P site (shown in pink) on the ribosome.
• In eukaryotes, initiator tRNA carries methionine (Met). (Bacteria use a modified methionine designated fMet.)

2. Elongation

• An aminoacyl-tRNA (a tRNA covalently bound to its amino acid) able to base pair with the next codon on the mRNA arrives at the A site (green) associated with:
  • an elongation factor (called EF-Tu in bacteria)
  • GTP (the source of the needed energy)
• The preceding amino acid (Met at the start of translation) is covalently linked to the incoming amino acid with a peptide bond (shown in red).
• The initiator tRNA is released from the P site.
• The ribosome moves one codon downstream.
• This shifts the more recently-arrived tRNA, with its attached peptide, to the P site and opens the A site for the arrival of a new aminoacyl-tRNA.
• This last step is promoted by another protein elongation factor (named EF-G) and the energy of another molecule of GTP.

Note: the initiator tRNA is the only member of the tRNA family that can bind directly to the P site. The P site is so-named because, with the exception of initiator tRNA, it binds only to a peptidyl-tRNA molecule; that is, a tRNA with the growing peptide attached.

The A site is so-named because it binds only to the incoming aminoacyl-tRNA; that is the tRNA bringing the next amino acid. So, for example, the tRNA that brings Met into the interior of the polypeptide can bind only to the A site.

3. Termination

• The end of translation occurs when the ribosome reaches one or more STOP codons (UAA, UAG, UGA). (The nucleotides from this point to the poly(A) tail make up the 3'-untranslated region [3'-UTR] of the mRNA.)
• There are no tRNA molecules with anticodons for STOP codons.

  (With a few special exceptions: link to mitochondrial genes and to nonstandard amino acids.)

• However, a protein release factor recognizes these codons when they arrive at the A site.
• Binding of this protein releases the polypeptide from the ribosome.
• The ribosome splits into its subunits, which can later be reassembled for another round of protein synthesis.

Polysomes

Quality Control

• premature STOP codons. Translation of these produces a truncated protein that is probably ineffective and may be harmful. The problem is solved by Nonsense-Mediated mRNA Decay (NMD).
• no STOP codon. These produce "nonstop" transcripts. The problem is solved by Nonstop mRNA Decay.

Nonsense-Mediated mRNA Decay (NMD)

• mutations, especially frameshifts;
• RNA processing (intron removal) errors;
• as an inevitable consequence of creating antigen receptors on B cells and T cells. [Link to discussion.]

Mechanism

• During RNA processing within the nucleus, protein complexes are added at each spot where adjacent exons are spliced together. (These are important signals for exporting the mRNA to the cytoplasm.)
• In the cytoplasm, as the ribosome moves down the mRNA, these complexes are removed (and sent back to the nucleus for reuse).
• If the ribosome encounters a premature STOP codon, the final exon-exon tag(s) are not removed, and this marks the defective mRNA for destruction.

Nonstop mRNA Decay

Nonstop transcripts occur when there is no STOP codon in the message. As a result the ribosome is unable to recruit the release factors needed to leave the mRNA.

Mechanisms

Eukaryotes and prokaryotes handle the problem of no STOP codon differently.

• In eukaryotes, when the ribosome stalls at the end of the poly(A) tail, proteins are recruited to
  • release the ribosome for reuse and to
  • degrade the faulty message.
• In prokaryotes, a special RNA molecule — called tmRNA saves the day. It is called tmRNA because it has the properties of both a transfer RNA and a messenger RNA.
  • The transfer part adds alanine to the A site on the ribosome.
  • The ribosome then moves on to the messenger part which encodes 10 amino acids that target the molecule for destruction (and releases the ribosome for reuse).

Regulation of Translation

The expression of most genes is controlled at the level of their transcription. Transcription factors (proteins) bind to promoters and enhancers turning on (or off) the genes they control.

However, there are some cases where gene expression is controlled at the level of translation instead.

By RNA Interference

• prevent it from being translated by ribosomes or
• trigger its destruction.

By Riboswitches

In many bacteria (and some eukaryotes), it turns out that the regulation of the level of certain metabolites is controlled by riboswitches. A riboswitch is a part of a molecule of messenger RNA (mRNA) with a specific binding site for the metabolite (or a close relative).

• If thiamine pyrophosphate (the active form of thiamine [vitamin B₁]) is available in the culture medium of E. coli,
  • it binds to a messenger RNA whose protein product is an enzyme needed to synthesize thiamine from the ingredients in minimal medium.
  • Binding induces an allosteric shift in the structure of the mRNA so that it can no longer bind to a ribosome and thus cannot be translated into the enzyme.
  • E. coli no longer wastes resources on synthesizing a vitamin that is available preformed.
• If vitamin B₁₂ is present in the cell,
  • it binds to the mRNA which encodes a protein needed to import the vitamin from the culture medium.
  • This, too, induces an allosteric shift in the mRNA that prevents it from binding a ribosome.
  • E. coli no longer wastes resources on synthesizing a transporter for a vitamin that it already has enough of.
• Some gram-positive bacteria (E. coli is gram-negative) control the level of a sugar needed to synthesize their cell wall with a riboswitch. In this case, as the concentration of the sugar builds up, it binds to the messenger RNA (mRNA) whose product is the enzyme that makes the sugar. This causes the mRNA to self-destruct so production of the enzyme — and thus the sugar — ceases.

Other riboswitches act on transcription rather than translation. [Link]

It has been suggested that these regulatory mechanisms, which do not involve any protein, are a relict from an "RNA world".

By Proteins

Translation of at least one mRNA in humans is repressed by a protein — aminoacyl tRNA synthetase. In response to the inflammatory cytokine interferon-gamma [IFN-γ], the synthetase abandons its normal function (adding Glu and Pro to their respective tRNAs) and instead binds to the mRNA blocking its translation.

In some bacteria, a protein product may inhibit the further translation of its own mRNA (a kind of feedback inhibition). It does so by binding to a site which blocks the mRNA from further association with a ribosome.

Summary

• transcription of the information encoded in DNA into a molecule of RNA (discussed in Gene Expression: Transcription) and
• translation of the information encoded in the nucleotides of mRNA into a defined sequence of amino acids in a protein (discussed here).

In eukaryotes, the processes of transcription and translation are separated both spatially and in time. Transcription of DNA into mRNA occurs in the nucleus. Translation of mRNA into polypeptides occurs on polysomes in the cytoplasm.

In prokaryotes (which have no nucleus), both these steps of gene expression occur simultaneously: the nascent mRNA molecule begins to be translated even before its transcription from DNA is complete.