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

into the A site first, where its anticodon is tested for complementarity to the mRNA codon present at that site. If the anticodon is complementary to the codon, the tRNA is retained; if the anticodon does not match the codon, the tRNA is rejected, and a new aa-tRNA is brought into the A site. A match between the anticodon and the codon in the A site triggers the tRNA in the A site to interact with the tRNA already present in the P site. The P site tRNA carries the growing polypeptide chain, and the next step is transfer of the growing peptide chain from the P site tRNA to the A site tRNA. The amino acid carried on the A site tRNA is attached to the carboxyl end of the polypeptide, and the polypeptide (now 1 amino acid longer) is now linked to the A site tRNA. The now unattached P site tRNA moves to the E site of the ribosome and the A site tRNA (which now carries the polypeptide) moves to the P site of the ribosome, which results in an empty A site. Each tRNA retains contact with the mRNA through anticodon-codon pairing so that the mRNA moves through the ribosome in concert with the tRNAs. This results in placement of the next codon of the mRNA in the empty A site, which is now available for entry of another aa-tRNA. Movement of the unattached P site tRNA into the E site results in ejection of the previous unattached tRNA from the E site, which allows the next tRNA to enter the cycle. The details of this process are described below.

Figure 2.22 Crystal structures of a tRNA and the ribosome. (A) Structure of a tRNA. The anticodon loop is at the bottom, and the 3′ acceptor end, where the amino acid or growing polypeptide is attached, is at the top. (From Yusupov MM, Yusupova GZ, Baucom A, et al, Science 292:883–896, 2001. Modified with permission from AAaS.) (B) The two subunits of the ribosome separated and rotated to show the channel between them through which the tRNAs move. The 30S subunit is on the left, and the 50S subunit is on the right. The tRNAs bound at the A, P, and E sites are indicated in yellow, green, and purple, respectively. (From Cate JH, Yusupov MM, Yusupova G, et al, Science 285:2095–2014, 1999. Modified with permission from AAAS.)

Details of Protein Synthesis

      In this section, we discuss the process of translation in more detail. First, we discuss reading frames, which determine which nucleotides in an mRNA are recognized as codons, and tRNA aminoacylation, which is responsible for correct matching of a tRNA with its cognate amino acid. Then, we discuss translation initiation, or how the 30S subunit finds the starting point of a coding sequence in the mRNA. Next, we discuss translation elongation, or what happens as the 70S ribosome moves along the mRNA, translating the information in the mRNA in the form of nucleotides into amino acids. Finally, we discuss how translation is terminated.

      READING FRAMES

      Each 3-nucleotide sequence, or codon, in the mRNA encodes a specific amino acid, and the assignment of the codons is known as the genetic code (Table 2.2). Because there are 3 nucleotides in each codon, an mRNA can be translated in three different frames in each region. Initiation of translation at a specific initiation codon establishes the reading frame of translation, so that in most cases only a single reading frame is utilized. Once translation has begun, the ribosome moves 3 nucleotides at a time through the coding part of the mRNA. If the translation is occurring in the proper frame for protein synthesis, we say the translation is in the zero frame for that protein. If translation is occurring in the wrong reading frame, it can be displaced either back by 1 nucleotide in each codon (the –1 frame) or forward by 1 nucleotide (the +1 frame). In a few instances, translational frameshifts that change the reading frame even after translation has initiated can occur. Frameshift mutations (see chapter 3) cause incorrect reading of an mRNA because of the insertion or deletion of nucleotides in the DNA sequence, which is then copied into mRNA. Translational frameshifting occurs when the ribosome shifts its position on the mRNA without a change in the mRNA sequence itself.

      TRNA AMINOACYLATION

Before translation can begin, a specific amino acid is attached to each tRNA by its cognate aaRS (Figure 2.24). Each of these enzymes specifically recognizes only one amino acid and one class of tRNA—hence the name cognate. How each aaRS recognizes its own tRNA varies, but the anticodon (i.e., the three tRNA nucleotides that base pair with the complementary mRNA sequence [Figure 2.25]) is not the only determinant. Other variations in the tRNA structure, such as the identity of the discriminator base immediately upstream of the CCA at the 3′ end (Figure 2.17) and the size of the variable loop, also contribute to aaRS recognition specificity. In some cases, if the anticodon in a given tRNA is mutated, the cognate aaRS still attaches the amino acid for the original tRNA, and that amino acid is inserted at a different codon in the mRNA. This is the basis of nonsense suppression, which is discussed in chapter 3. The amino acid is attached to the terminal A residue on the tRNA, and the energy for the reaction is provided by cleavage of ATP. Finally, the tRNA with its amino acid is bound to the protein EF-Tu, which assists in delivery of the aa-tRNA to the ribosome.

Figure 2.23 Overview of translation. (A) The ribosomal A (aminoacyl) site is empty, the growing peptide chain is attached to the P (peptidyl) site tRNA, and the tRNA that previously contained the peptide chain is in the E (exit) site. (B) The tRNA bound to its amino acid and complexed with elongation factor Tu (EF-Tu) and guanosine triphosphate (GTP) comes into the empty A site and remains there if its anticodon matches the mRNA codon at that site; the E site tRNA leaves the ribosome. EF-Tu–GTP is converted to EF-Tu–GDP and released from the ribosome, then is recycled to EF-Tu–GTP by EF-Ts. (C) Peptidyltransferase (23S rRNA in the 50S ribosome) catalyzes peptide bond formation between the carboxyl end of the growing polypeptide carried by the P site tRNA and the amino end of the amino acid carried by the A site tRNA. (D) Translation elongation factor G (EF-G) catalyzes translocation of the A site tRNA to the P site, making room at the A site for another aminoacyl-tRNA. The previous P site tRNA, now stripped of its polypeptide, moves to the E site before exiting the ribosome.

      Table 2.2 The genetic code

First position (5′ end)

Second position

Third Скачать книгу В начало < 52 53 54 55 56 57 58 59 60 61 > В конец