Control Of Translational Accuracy Research Articles

The control of translational accuracy is a determinant of healthy ageing in yeast.

Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.

Positions 13 and 914 in Escherichia coli 16S ribosomal RNA are involved in the control of translational accuracy.

Using a conditional expression system with the temperature-inducible lambda PL promoter, we previously showed that the single mutations 13U-->A and 914A-->U, and the double mutation 13U-->A and 914A-->U in Escherichia coli 16S ribosomal RNA impair the binding of streptomycin (Pinard et al., The FASEB Journal, 1993, 7, 173-176). In this study, we found that the two single mutations and the double mutation increase translational fidelity, reducing in vivo readthrough of nonsense codons and frameshifting, and decreasing in vitro misincorporation in a poly(U)-directed system. Using oligodeoxyribonucleotide probes which hybridize to the 530 loop and to the 1400 region of 16S rRNA, two regions involved in the control of tRNA binding to the A site, we observed that the mutations in rRNA increase the binding of the probe to the 530 loop but not to the 1400 region. We suggest that the mutations at positions 13 and 914 of 16S rRNA induce a conformational rearrangement in the 530 loop, which contributes to the increased accuracy of the ribosome.

Interaction of the yeast omnipotent suppressors SUP1(SUP45) and SUP2(SUP35) with non-mendelian factors.

The SUP1 and SUP2 genes code for protein factors intimately involved in the control of translational accuracy. The disrupted alleles of these genes confer a recessive lethal phenotype in both [psi+] and [psi-] genetic backgrounds, indicating an essential function for the corresponding proteins. In [psi+] diploids, heterozygous for the SUP1 null allele, several dominant phenotypes were evident with slow growth and inability to sporulate. These dominant phenotypes disappear after transformation with the multicopy plasmid carrying the wild-type allele of the SUP1 gene. Such dominant phenotypes were not observed for the SUP2 null allele. The incompatibility of multicopy plasmids carrying the SUP2 gene with guanidine hydrochloride-curable cytoplasmic factor(s) was also demonstrated. The possible mechanisms of interaction of the SUP1 and SUP2 genes with the [psi] determinant are discussed.

Divergence and conservation of SUP2(SUP35) gene of yeasts Pichia pinus and Saccharomyces cerevisiae

SUP2 (SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part and a region homologous to EF-1 alpha. The comparison of amino acid sequences of the Sup2 proteins reveals high conservation (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae were found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.

A deletion mutation at the 5′ end of Escherichia coli 16S ribosomal RNA

A deletion of five nucleotides was introduced at the 5′ end of the Escherichia coli 16S rRNA gene cloned in an appropriate vector under control of a T7 promoter. The 16S rRNA generated by in vitro transcription could be assembled into 30S subunits. The deletion did not affect the efficiency of translation of natural messengers and the correct selection of the reading frame. However, it reduced the binding of the messengers, which suggests that the 5′ end of 16S rRNA is located on the pathway followed by the messengers on the 30S subunits. The deletion also restricted the stimulation of misreading by streptomycin in a poly(U)-directed system. This is in accord with the proximity of the 5′ end of 16S rRNA to proteins S4, S5 and S12, which are known to be involved in the control of translational accuracy.

Antagonistic effects of mutant elongation factor Tu and ribosomal protein S12 on control of translational accuracy, suppression and cellular growth

Kirromycin-resistant mutant forms of elongation factor Tu, which are coded by tufA (Ar) or tufB (Bo) and are associated with an increased rate of translational error formation, have been analysed. In vivo, Ar was found to increase misreading as well as suppression of non-sense codons irrespective of Bo in a strain with wild type ribosomes. It is therefore not necessary to evoke both tufA (Ar) and tufB (Bo) mutations together in order to increase translational error as suggested earlier [1]. When combined with a hyperaccurate ribosomal rpsL (S12) mutation, Ar counteracts the restrictive effects on translational error formation caused by the altered protein S12, thus restoring the levels of missense error in vitro non-sense error and suppression in vivo to near wild type values. As judged from in vitro experiments this results principally from a lowered selectivity of the Ar ternary complex at the initial discrimination step on the ribosome during translation. In vivo, this compensatory effect on the rpsl mutation on non-sense error formation and suppression is seen irrespective of the nature of tRNA or codon context. Furthermore, the tufA mutation enhances the cellular growth rate of the rpsL mutant, whereas it decreases growth of strains with normal ribosomes. Inactivation of one of the two genes coding for EF-Tu ( tufB), while leaving the other gene ( tufA) intact, can by itself, increase non-sense error formation and suppression.

At least seven ribosomal proteins are involved in the control of translational accuracy in a eukaryotic organism

In the filamentous fungus Podospora anserina, ribosomal proteins of 60 mutants impaired in the control of translational fidelity have been submitted to electrophoretic analysis. The “four corners” system combining four different two-dimensional polyacrylamide gel electrophoretic systems has been used. An altered electrophoretic pattern has been observed for 12 mutants. In mutants su3, su12 and su11 (decreased translational fidelity), proteins S1, S7 and S8, respectively, are altered. For AS mutants (increased translational fidelity), proteins S9, S12 and S19, respectively, are altered in AS9, AS1 and AS6 mutants, and protein S29 is lacking in AS3 mutants. The data suggest that five of these genes (at least) are the structural genes for the relevant proteins ( su3:S1, su12:S7, AS1:S12, AS6:S19, AS9:S9), while the AS3 gene may code for a modifying enzyme.

Identification of the structural gene for the S9 ribosomal protein in the fungus Podospora anserina: a new protein involved in the control of translational accuracy.

AS9-1 was isolated as a mutation restoring growth in a strain carrying the ribosomal mutation su12-1. The AS9-1 mutation confers a weak antisuppressor effect and a low level of resistance to paromomycin. Two-dimensional polyacrylamide gel electrophoresis patterns of the ribosomal proteins from AS9-1 strains show an altered S9 protein which is more basic than the wild-type form. The presence of the two forms of the protein (wild-type and mutant) in heterocaryotic strains strongly suggests that AS9 is the structural gene for the ribosomal protein S9.

Involvement of ribosomal protein L7/L12 in control of translational accuracy.

The effects of two mutations, which map at the rplL locus and both give a changed 50S ribosomal protein L7/L12, were studied. Both mutations are associated with an increased misreading of all three nonsense codons in vivo and ribosomes from the mutants give an increased misreading of the phenylalanine codon UUU by tRNALeu in vitro. The rplL-associated misreading in vitro is not limited to a particular type of mRNA or tRNA. Results from a translational proofreading assay, using mutant ribosomes, suggest that protein L7/L12 is involved in the control of translational accuracy by contributing to the efficiency of a translational proofreading step(s).