Fidelity of translation

There is one separate tRNA for each amino acid and one separate specific synthetase. The enzyme must bind not only the correct amino acid but also the correct tRNA, so that each synthetase has specific recognition sites for both. Transfer RNAs contain a three-base sequence that is an anticodon, which binds to its complementary codon on messenger RNA. The importance of the synthetase in relation to fidelity of translating the information in messenger RNA is indicated by the fact that, once an amino acid is bound to tRNA, its identity as an amino acid is dictated by the anticodon site on the transfer RNA and not by the amino acid itself. (The enzyme can be considered as a dictionary, since it provides a cross-reference between the nucleic acid and amino acid languages.)... 

That aminoglycoside binding interferes with translation has been known for at least four decades. The mode of action of these antibiotics decreases the fidelity of translation, such that the organism biosynthesizes proteins that are defective. The presence of defective or nonfunctional proteins leads to the demise of bacteria. Consequently, paromomycin s bactericidal mode of action may come from its ability to lock the A-site into a conformation in which discrimination of cognate and near cognate tRNA is significantly diminished. 

Mutants in ribosomal RNA that affect the fidelity of translation termination... 

The aminoglycosides decrease the fidelity of translation by binding to the 30S subunit of the ribosome. This permits the formation of the peptide initiation complex but prohibits any subsequent addition of amino acids to the peptide. This effect is due to the inhibition of polymerization as well as to the failure of tRNA and mRNA codon recognition. Aminoglycosides are ototoxic (i.e., may produce partial deafness), damaging the auditory nerve. Kanamycin is less toxic. Since aminoglycosides are concentrated in the kidney, they may occasionally cause kidney damage. 

After many such elongation cycles, synthesis of the polypeptide is terminated with the aid of release factors. At least four high-energy phosphate equivalents (from ATP and GTP) are required to generate each peptide bond, an energy investment required to guarantee fidelity of translation. 

In vitro, the fidelity of translation is strongly influenced by the concentration of Mg2 + ions in the reaction. In the range from 1 to 4 mM Mg2 +, the ribosome will require a Shine-Delgarno sequence (prokaryotic) or a 7-methyl-guanosine cap (eukaryotic) on the mRNA before translation will initiate. For this reason, in vitro translation experiments performed with naturally occurring mRNAs are most... 

There are four variables in the general in vitro translation protocol that you will investigate. First, you will vary the Mg2 + concentration to study its effect on the fidelity of translation initiation. Second, you will investigate the specificity of the 3H-Phe-tRNA and the mRNA codon with which it is able to interact. Third, you will study the effects of different ribonuclease enzymes on the various RNA components present in the reaction (see below). Finally, you will investigate the ability of the ribosome to carry out translation in the presence of three different inhibitors with different modes of action (see below). 

Why is the fidelity of translation initiation in vitro dependent on the concentration ofMg2 + ... 

From these data, what specifics can you conclude about the microbe s genetic code What is the sequence of the anticodon loop of a tRNA carrying a threonine If you found that this microbe contained 61 different tRNAs, what could you speculate about the fidelity of translation in this organism ... 

Binds to the A site and prevents entry of aminoacyl tRNA Inhibits the 50S ribosome peptidyl transferase activity Inhibits the fidelity of translational initiation Inactivates eukaryotic elongation factor eEF-2 Inactivates 28S rRNA by A-glycolytic cleavage of an adenine Inhibits the 60S ribosome peptidyl transferase activity... 

The result is the immediate termination of translation and the release of a truncated protein. Two potent antibiotics that specifically inhibit bacterial translation, are tetracycline, which blocks the A site and prevents the entry of aminoacyl-tRNAs, and chloramphenicol, which inhibits the peptidyl transferase activity of the 23 S rRNA. The mechanisms of action of these antibiotics, including streptomycin, which alters the fidelity of translation in bacteria, are listed in Table 26.1. 

The same is true of bulge loop SOS-SIO and the loop sequence S18-S33, which contains 7-methylguanine (m G) at position S26. Reconstitution experiments also suggested that S16 binds to S4 as well as to S20. Some mutations in proteins S4 and S5 are associated with reduced fidelity of translation, while others lead to spectinomycin resistance. 

The recognition of the correct tRNA by the synthetase is vital to the fidelity of translation because most of the final proofreading occurs at this step. See the articles by LaRiviere et al. and Ibba cited in the bibliography at the end of this chapter for the latest information on this topic. 

In the last step of the gene expression pathway, genomic information encoded in messenger RNAs is translated into protein by a ribonucleoprotein called the ribosome. The prokaryotic ribosome (MW approximately 2.6 x 10 daltons) is about 2/3 RNA and 1/3 protein and consists of 2 subunits the larger of which is approximately twice the molecular weight of the smaller. The small subunit mediates the interaction between the mRNA codon and the tRNA anticodon, an interaction on which the fidelity of translation depends. The large subunit, which sediments at SOS in prokaryotes, includes the activity that catalyzes peptide bond formation - peptidyl transferase. 

The important feature of this methodology is that fidelity of the amide bond formation is achieved by specific and template-directed coupling of the desired acyl transfer. Therefore, proper juxtaposition of peptidyl- and aminoacyl-oligonucleotides on a polynucleotide template control the direction of polypeptide synthesis. This is essentially the way that fidelity of translation in protein biosynthesis is taking place in natural systems. 

Initiation Factors 2 and 3 have seemingly opposing functions. While IF2 promotes the binding of tRNA to the 308 subunit, IF3 can be considered the subunit antiassociation factor because it increases the rates of subunit exchange and complex dissociation. In fact the two functions cooperate to facilitate formation of the correct 308 initiation complex— IF2 preferentially enhances the binding of the amino-blocked initiator tRNA and IF3 specifically increases the rate of noninitiator tRNA dissociation from the ternary complex. Initiation Factor 3 also contributes to the fidelity of translation by confirming the codon-anticodon interaction on the 308 subunit. 

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