Releasing factors prokaryotic

Competition with release factors can be reduced by deactivating a release factor in a cell-free lysate prepared from a prokaryote. This option is not available in rabbit reticulyte lysate since mammals have only two release factors (RFs), termed eukaryotic release factors 1 and 3 (eRFl and eRF3). In eukaryotes, eRFl recognizes all three stop codons and eRF3 stimulates eRFl activity in the presence of GTP [41]. Deactivation of either RF would effectively deactivate all three stop codons, and at least one termination codon must function properly for the suppression method to be useful. 

Abbreviations aa-tRNA Amino-acyl tRNA eLF Eukaryotic translation initiation factor IF Prokaryotic translation initiation factor eEF Eukaryotic translation elongation factor EF Prokaryotic translation elongation factor eRF Eukaryotic translation termination factor (release factor) RF Prokaryotic translation release factor RRF Ribosome recycling factor Rps Protein of the prokaryotic small ribosomal subunit Rpl Protein of the eukaryotic large ribosomal subunit S Protein of the prokaryotic small ribosomal subunit L Protein of the prokaryotic large ribosomal subunit PTC Peptidyl transferase center RNC Ribosome-nascent chain-mRNA complex ram Ribosomal ambiguity mutation RAC Ribosome-associated complex NMD Nonsense-mediated mRNA decay... 

Eventually, one of three termination codons (also called Stop codons) becomes positioned in the A site (Fig. 7). These are UAG, UAA and UGA. Unlike other codons, prokaryotic cells do not contain aminoacyl-tRNAs complementary to Stop codons. Instead, one of two release factors (RF1 and RF2) binds instead. RF1 recognizes UAA and UAG whereas RF2 recognizes UGA. A third release factor, RF3, is also needed to assist RF1 or RF2. Thus either RF1 + RF3 or RF2 + RF3 bind depending on the exact termination codon in the A site. RF1 (or RF2) binds at or near the A site whereas RF3/GTP binds elsewhere on the ribosome. The release factors cause the peptidyl transferase to transfer the polypeptide to a water molecule instead of to aminoacyl-tRNA, effectively cleaving the bond between the polypeptide and tRNA in the P site. The polypeptide, now leaves the ribosome, followed by the mRNA and free tRNA, and the ribosome dissociates into 30S and 50S subunits ready to start translation afresh. 

The coding region ends with the protein sequence—that is, there is no stop codon present. In the prokaryotic system the presence of a stop codon would result in the binding of the release factors (Grentzmann et al, 1995 Tuite and Stansfield, 1994) and the ribosome recycling factor (Janosi et al., 1994) to the mRNA-ribosome-protein complexes. This would then lead to the release of the protein by hydrolysis of the peptidyl-tRNA (Tate and Brown, 1992), thereby dissociating the ribosomal complexes (Fig. 4A). A similar mechanism exists in eukaryotic systems (Frolova et al., 1994 Zhouravleva et al., 1995). 

The elongation reaction described earlier for the prokaryotic system is essentially the same as that found in the eukaryotic system. The final difference between the two systems lies in the mechanism of translational termination. Recall that the prokaryotic system utilizes two different release factor proteins. The eukaryotic system relies on a single release factor protein, eRF. 

The structure resembles that of a tRNA by molecular mimicry. The sequence Gly-Gly-Gln, present in both eukaryotes and prokaryotes, occurs at the end of the structure corresponding to the acceptor stem of a tRNA. This region binds a water molecule. Disguised as an aminoacyl-tRNA, the release factor may carry this water molecule into the peptidyl transferase center and, assisted by the catalytic apparatus of the ribosome, promote this water molecule s attack on the ester linkage, freeing the polypeptide chain. The detached polypeptide leaves the ribosome. Transfer RNA and messenger RNA remain briefly attached to the 70S ribosome until the entire complex is dissociated in a GTP-dependent fashion by ribosome release factor (RRF) and EF-G. Ribosome release factor is an essential factor for prokaryotic translation. 

Elongation and termination. Eukaryotic elongation factors EFla and EFip y are the counterparts of prokaryotic EF-Tu and EF-Ts. The GTP form of EFla delivers aminoacyl-tRNA to the A site of the ribosome, and EFip y catalyzes the exchange of GTP for bound GDP. Eukaryotic EF2 mediates GTP-driven translocation in much the same way as does prokaryotic EF-G. Termination in eukaryotes is carried out by a single release factor, eRFl, compared with two in prokaryotes. Finally, eIF3, like its prokaryotic counterpart IF3, prevents the reassociation of ribosomal subunits in the absence of an initiation complex. 

Figure 2 In ribosome display, mRNA (A) extracted from a cell is converted into a cDNA library (B) is transcribed back into mRNA with no stop codons. Prokaryotic or eukaryotic proteosomes are added and the ribosome then travels down the mRNA (C) translating until it reaches the end of the mRNA molecule (D), where the ribosome halts. With no stop codon, the release factor proteins cannot bind and so the protein, ribosome, and mRNA are physically associated and can be stabilized by high Mg2+ and low temperatures. This complex could then be bound directly to an immobilized natural product (E), the nonbinding library members washed away and the bound members eluted with EDTA (F), which destabilizes the ribosomal complexes by removing Mg2+. The purified sublibrary is converted into cDNA by reverse transcription (RT-PCR) and amplified by regular PCR (B). The/n vitro transcription and translation can be repeated for another round of selection or the cDNA can be analyzed by agarose electrophoresis and/or sequencing.

As in prokaryotic termination, the ribosome encounters a stop codon, either UAG, UAA, or UGA, and these are not recognized by a tRNA molecule. In prokaryotes, three different release factors—RFl, RF2, and RF3—were used. 

Eukaryotic chain elongation is similar to the prokaryotic counterpart. With chain termination, there is only one release factor that binds to all three stop codons. 

The critical role that protein factors play in translation is discussed next, including initiation, elongation, and release factors. The termination of translation is outlined, and the role of release factors that recognize translation stop codons is described. The chapter closes with a brief overview of translation in eukaryotes, emphasizing the major contrasting features with respect to translation in prokaryotes. Differences in the initiator tRNA, the selection mechanism of the initiator codon, the ribosomes, and the overall complexity of the process are highlighted. Last, the mechanisms of several potent inhibitors of translation and the mechanism of the bacterial toxin that causes diphtheria is presented. 

Termination in eukaryotes resembles that in prokaryotes but requires only a single release factor, eRF, which binds to the ribosome together with GTP. The hydrolysis of GTP triggers the dissociation of eRF form the ribosome. 

In prokaryotes, two release factors have been identified, one (RFl) recognizing UAA and UAG, the other (RF2) functioning with UGA. Ribosomal binding and release of RFl and RF2 are stimulated by a third factor, RF3, which interacts with GTP and GDP. In eukaryotic cells such as reticulocytes, one release factor (eRF) has been found to function with all three termination codons, and the binding of this factor to ribosomes is stimulated by GTP but not GDP. Although the details are not entirely clear, GTP hydrolysis appears to be required for the release of the finished polypeptide chain by cleavage of the peptidyl-tRNA bond and completion of the termination process leading to dissociation of the release factor from the ribosome. 

The observation that prokaryotic RFl and RF2 exhibit specificity for different RNA sequences suggests that direct contacts are made between the release factors and the mRNA. These protein-RNA interactions contrast with the codon-anticodon contacts defined by Watson-Crick basepairing rules, and hint at a mechanism in which the proteins mimic bound tRNA. 

There is a single prokaryotic RNA polymerase that synthesizes all types of RNA in the cell. The core polymerase responsible for making the RNA molecule has the subunit structure Ojpp. A protein factor called sigma (a) is required for the initiation of transcription at a promoter. Sigma factor is released immediately after initiation of transcription. Termination of transcription sometimes requires a protein called rho (p) faaor. This enzyme is inhibited by rifampin. Actinomycin D binds to the DNA preventing transcription. 

The first phase of translation, initiation, involves several steps. First, two proteins, initiation factors IF-1 and IF-3, bind to the 30 S subunit (1). Another factor, IF-2, binds as a complex with GTP (2). This allows the subunit to associate with the mRNA and makes it possible for a special tRNA to bind to the start codon (3). In prokaryotes, this starter tRNA carries the substituted amino acid N-formylmethionine (fMet). In eukaryotes, it carries an unsubstituted methionine. Finally, the 50 S subunit binds to the above complex (4). During steps 3 and 4, the initiation factors are released again, and the GTP bound to IF-2 is hydrolyzed to GDP and Pj. 

Describe the role of sigma factors (o) in transcription by prokaryotic RNA polymerases. What is the effect of the release of o from the holoenzyme once transcription has been initiated. How would a mutation that prevents a o factor from dissociating from core RNA polymerase affect the rate of transcription ... 

E. coli has three initiation factors (fig. 29.13) bound to a small pool of 30S ribosomal subunits. One of these factors, IF-3, serves to hold the 30S and 50S subunits apart after termination of a previous round of protein synthesis. The other two factors IF-1 and IF-2, promote the binding of both fMet-tRNA,Mel and mRNA to the 30S subunit. As we noted before, the binding of mRNA occurs so that its Shine-Dalgarno sequence pairs with 16S RNA and the initiating AUG sequence with the anticodon of the initiator tRNA. The 30S subunit and its associated factors can bind fMet-tRNAjMet and mRNA in either order. Once these ligands are found, IF-3 dissociates from the 30S, permitting the 50S to join the complex. This releases the remaining initiation factors and hydrolyzes the GTP that is bound to IF-2. The initiation step in prokaryotes requires the hydrolysis of one equivalent of GTP to GDP and Pj. 

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