Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin

Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin


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formylmethionine for initiation of translation. In contrast, eukaryotes do not seem to use special sequence elements and instead usually use the first AUG from the 5′ end of the mRNA as the initiation codon. Sequences around this initiator AUG may also be important for its recognition, and secondary structure in the mRNA may mask other AUG sequences that could potentially be used as initiator codons. Although eukaryotes have a special methionyl tRNA that responds to the first AUG codon, called Met-tRNAi, the methionine attached to the eukaryotic initiator tRNA is never formylated. As in bacteria, however, the first methionine is usually removed by an aminopeptidase after the protein is synthesized. Eukaryotes and archaea also seem to use many more initiation factors and elongation factors than do bacteria. The archaea use formylated methionine and S-D sequences like bacteria, but their initiation factors are more similar to those in eukaryotes. Although the exact roles of most of these initiation factors are unknown in archaea, many are obviously related to the initiation and translation factors of bacteria. It therefore appears that the mechanism of translation initiation in the archaea is a sort of hybrid between that in bacteria and that in eukaryotes.

      TRANSLATION ELONGATION

      During translation elongation, the ribosome moves 3 nucleotides at a time along the mRNA in the 5′-to-3′ direction, allowing tRNAs carrying amino acids (aa-tRNAs) to pair with the mRNA. Each tRNA has a specific 3-nucleotide anticodon sequence in one of its loops, and these nucleotides must be complementary to the mRNA codon for the tRNA to be bound tightly to the ribosome (Figure 2.25). As in other nucleic acids, pairing is antiparallel, so that the two RNA sequences are complementary when read in opposite directions. In other words, the 3′-to-5′ sequence of the anticodon must be complementary to the 5′-to-3′ sequence of the codon.

      Entry of aa-tRNAs bound by EF-Tu into the ribosome is random. If the anticodon is complementary to the mRNA codon at the A site (Figure 2.23), codon-anticodon pairing stimulates a structural transition of the ribosome that promotes the next step. A mispaired tRNA is not stabilized and is released from the ribosome. This pairing of only three bases is sufficient to direct the correct tRNA to the A site on the ribosome; in fact, sometimes the pairing of only two bases is sufficient to direct the anticodon-codon interaction (see “Wobble” below). Accurate codon-anticodon pairing is monitored by specific residues in 16S rRNA that form the decoding site. The tRNA is positioned across the 30S and 50S subunits of the ribosome so that the anticodon loop is in communication with the mRNA in the 30S subunit and the acceptor end of the aa-tRNA containing the bound amino acid is in communication with the 23S rRNA in the 50S subunit. The conformational change in the ribosome that is triggered by accurate codon-anticodon pairing results in hydrolysis of the GTP on EF-Tu to GDP and release of EF-Tu–GDP from the ribosome. EF-Tu–GDP is recycled into EF-Tu–GTP by the action of another protein factor, EF-Ts (Figure 2.23).

      After peptide bond formation, the P site tRNA no longer has anything attached to it, while the A site tRNA carries the polypeptide chain. Another enzyme, translation elongation factor G (EF-G), in complex with GTP, then enters the ribosome and moves (or translocates) both the polypeptide-containing tRNA and the mRNA from the A site to the P site, moving the deacylated tRNA from the P site to the E site and making room for another aa-tRNA to enter the A site. Translocation requires hydrolysis of the GTP bound to EF-G, which also results in release of EF-G from the ribosome (Figure 2.23). The deacylated tRNA in the E site later exits the ribosome (possibly in conjunction with entry of the next aminoacyl-tRNA into the A site). The mRNA maintains contact with the tRNAs as they move through the ribosome and therefore also moves through the ribosome 3 nucleotides at a time.

Schematic illustration of the peptidyltransferase reaction catalyzes dissociation of the carboxyl end of the formyl-methionine from the P site tRNAfMet and peptide bond formation with the amino end of the amino acid on the A site tRNA.

      TRANSLATION TERMINATION

      During elongation, translation proceeds along the mRNA, one codon at a time, until the ribosome encounters one of three special codons, UAA, UAG, or UGA, that serve as translational stop signals. These codons, designated termination codons, stop codons, or nonsense codons (because they do not encode an amino acid), have no corresponding tRNA (Table 2.2). When a termination codon enters the A site of a translating ribosome, translation stops because no aa-tRNA can match the codon. Similar to the positioning of translation initiation codons, the termination codon that terminates translation may not be at the 3′ end of the mRNA molecule. The region between the termination codon and the 3′ end of the mRNA (or a downstream coding sequence for another polypeptide in a transcript that encodes multiple proteins [see below]) is called the 3′ untranslated region (3′-UTR).

      RELEASE FACTORS


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