TRNA translocation process

The ribosomal translocation process is quite complex. As the tRNAs move from A to P to E sites on the 16S RNA platform, the mRNA must also move in discrete single-codon steps. Tire acceptor stems of the tRNAs in the A and P sites must react at the appropriate times in the peptidyltransferase center. Study of protection from chemical probes suggests that tRNAs sometimes lie with the anticodon loop in the A site of the small ribosomal subunit, while the acceptor stem is in the P site of the large subunit (an A/P site as illustrated in Fig. 29-12B). Each aminoacyl-tRNA enters as a complex with EF-Tu and may initially bind with its anticodon in the A site and the acceptor stem with attached EF-Tu in a transient T site, the composite state being A/T. After loss of EF-Tu the acceptor stem can move into the A site to give an A/A state. The peptidyltransferase reaction itself necessarily involves movement at the acceptor stems by 0.1 nm or more. However, additional movement of 1 nm is needed to move the two tRNAs into states A/P and P/E, respectively. Movement of the mRNA then moves the... 

The final step of elongation is the translocation of the ribosome a distance of one codon in the 30 direction. This step requires the hydrolysis of another GTP and results in the displacement of the A site into the codon3 position. The dipeptidyl-tRNA stays bound to the mRNA at codon2, but since the ribosome is translocated, this tRNA is now within the P site. This translocation process also creates an empty A site for the next aminoacyl-tRNA and the E site (not shown in Fig. 26.11) now contains the deacylated-tRNA. The bacterial elongation factor EF-G is required for ribosome translocation and it is thought that hydrolysis of GTP results in a conformational change in the ribosome to facilitate the translocation step. This series of three steps is repeated for each amino acid added to the growing polypeptide chain (Fig. 26.13). 

The process of tRNA translocation occurs in two discrete steps (Figure 13.16) ... 

Zasloff. M. (1983). tRNA transport from the nucleus in a eukaryotic cell Carrier-mediated translocation process. Proc. Natl. Acad. Sci. U.S.A. 80(21), 6436-6440. 

When the 60 S ribosomal subunit joins the 40 S, the acceptor or A site is actually formed for the first time. Two proteins, EF-1 and EF-2, as well as GTP, are required for this process. EF-1 recognizes and binds all aminoacyl-tRNAs, save for the initiator tRNA, to the A site. EF-2 is involved in translocation of peptidyl-tRNA from the A site to the D or P site, in association with the coordinated movement of mRNA and the ejection of deacylated tRNA from the D or P site on the ribosome. Diphtheria toxin catalyzes an ADP-ribosylation of EF-2 in a reaction where NAD acts as the donor of the ADP-ribosyl moiety. As a consequence of this reaction, the EF-2 is rendered ineffective in the translocation process. The process of elongation has been reviewed by Miller and Weissbach (1977), Mazumder and Szer (1977), and Safer and Anderson (1978), and that of translocation by Brot (1977) and Spirin (1978). Both processes are depicted in Figs. 7 and 8. 

The energetics of the translocation process and the role of GTP therein have fascinated investigators for many years. Brot (1977) and Spirin (1978) have reviewed this process and the proposed means by which translocation is carried out. Spirin has suggested that, while the ribosome binds peptidyl-tRNA on the acceptor site (i.e., prior to translocation), the whole system is akin to a high-energy intermediate in which eventual translocation is thermodynamically ensured. This provocative hypothesis merits careful reading in view of its similarity to the role of the membrane in the chemiosmotic hypothesis for oxidative phosphorylation (Chapter 9). 

Elongation is a cycUc process on the ribosome in which one amino acid at a time is added to the nascent peptide chain. The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, and (3) translocation. 

The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

For processive peptide polymerization, the rihosome has to move along the mRNA. Following peptide bond formation, the rihosomal A site is occupied hy a peptidyl-tRNA whereas the P site contains a deacylated tRNA. During translocation, the complex of the two tRNAs with the mRNA has to move relative to the ribosome to... 

Then the peptidyl-tRNA at the A site is translocated to the P site by the ribosome moving along the mRNA a codon at a time, exposing the A site for a new aminoacyl-tRNA appropriate for the particular codon, and a repeat of the elongation process occurs. The cycles of elongation and translocation continue until a termination codon is reached, and the peptide or protein is then hydrolysed and released... 

Overview of reactions in protein synthesis. (aab aa2, aa3 = amino acids l, 2, 3.) Protein synthesis requires transfer RNAs for each amino acid, ribosomes, messenger RNA, and a number of dissociable protein factors in addition to ATP, GTP, and divalent cations. First the transfer RNAs become charged with amino acids, then the initiation complex is formed. Peptide synthesis does not start until the second aminoacyl tRNA becomes bound to the ribosome. Elongation reactions involve peptide bond formation, dissociation of the discharged tRNA, and translocation. The elongation process is repeated many times until the termination codon is reached. Termination is marked by the dissociation of the messenger RNA... 

The final step in elongation is known as translocation (fig. 29.17). This reaction, like aminoacyl-tRNA binding, is catalyzed by a factor (the translocation factor, known as EF-G in prokaryotic systems and EF-2 in eukaryotic systems) that cycles on and off the ribosome and hydrolyzes GTP in the process. The overall purpose of translocation is to move the ribosome physically along the mRNA to expose the next codon for translation. 

Protein synthesis is an energy-intensive process. High-energy phosphate bonds are expended for each peptide bond formed. One high-energy bond is consumed when an amino acid is activated by its aminoacyl-tRNA synthetase. Delivery of aminoacyl-tRNA by EF-Tu consumes one GTP per amino acid, and the translocation reaction consumes another. 

After the correct aminoacyl-tRNA has been placed in the A site, the transfer of the polypeptide chain from the tRNA in the P site is a spontaneous process, driven by the formation of the stronger peptide bond in place of the ester linkage. However, protein synthesis cannot continue without the translocation of the mRNA and the tRNAs within the ribosome. The mRNA must move by a distance of three nucleotides as the deacylated tRNA moves out of the P site into the E site on the 30S subunit and the peptidyl-tRNA moves out of the A site into the P site on the 30S subunit. The result is that the next codon is positioned in the A site for interaction with the incoming aminoacyl-tRNA. 

During elongation the protein is S3mthesized one amino acid at a time on the SOS ribosome. This process occurs in three major steps binding of charged tRNA, peptide bond formation, translocation of the growing peptide chain. 

P-site The peptidyl site on the ribosome to which Met-tRNA is brought to base pair with the mRNA sequence AUG. It is also the site to which the peptidyl RNA is moved in a process known as translocation following the formation of a new peptide bond. 

The second step in the elongation of protein synthesis is the peptidyl-transferase reaction in which a peptide bond is formed between the amino acid in the P site and the amino acid coupled to aminoacyl-tRNA in the A site. This reaction is facilitated by the intrinsic activity of the ribosome. As a result of this reaction, the growing polypeptide chain becomes elongated without moving forward. The movement or translocation of the dipeptide-tRNA from the A site to the P site is achieved by the action of EF-2, while another molecule of GTP is hydrolyzed. This process results in the relative movement of the mRNA by three nucleotides, so that a new codon becomes readable in the A site. The deacylated tRNA is pushed out of the ribosome after a transient halt at the so-called exit (E) site. At this point, all the components... 

When codon-anticodon base pairing occurs the amino acid attached to the tRNA is correctly positioned within the ribosome for peptide bond formation. As each peptide bond is formed, the newly incorporated amino acid is released from its tRNA and the mRNA moves relative to the ribosome so that a new codon enters the catalytic site. The latter process is called translocation. Translation continues one codon at a time until a special base sequence, called a termination or stop codon, is reached. The polypeptide is then released from the ribosome, and folds into its biologically active conformation. Depending on the type of polypeptide, it may then bind to other folded polypeptides to form larger complexes. 

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