Mutants of elongation factor Tu promote ribosomal frameshifting and nonsense readthrough.

This is the first report of ribosomal frameshifting promoted by mutants of the elongation factor Tu (EF-Tu). EF-Tu mutants can suppress both -1 and +1 frameshift mutations. The level of nonsense readthrough is also increased at some UGA (this paper) and UAG (Hughes, 1987) sites by these mutants. Suppression occurs when a mutant tuf allele is paired with a wild-type copy of the other tuf gene but is most efficient when both tuf genes are mutant. Frameshifting mediated by the tuf alleles studied, tufA8 and tufB103, is not general; indeed most frameshift mutations are not suppressed. Several possible mechanisms by which mutant EF-Tu may cause frameshifting are discussed.     

Error-prone EF-Tu reduces in vivo enzyme activity and cellular growth rate

Mutations in Salmonella typhimurium encoding error-prone EF-Tu (tufA8, tufB103) enhance translational error levels and also cause a reduced growth rate. The relative changes in error level and growth rate are inversely related and dependent on the status of the two tuf genes. Possible causes of the reduced growth rate were investigated. Several important parameters with the potential to alter growth rate (the EF-Tu-ribosome interaction, the in vivo elongation rate and the processivity of translation), are all relatively unaffected by the tuf mutations. The small reduction in processivity observed in some strains is not quantitatively related to the growth rate reduction. Instead, the error-enhancing mutations are associated with a large reduction in the specific activity of a test protein, beta-galactosidase, suggesting by inference that the reduced growth rate is a consequence of the synthesis of error-containing proteins.     

Mutant forms of tufA and tufB independently suppress nonsense mutations

The level of nonsense suppression in Salmonella typhimurium carrying error-enhancing mutations in either or both of the genes coding for the elongation factor EF-Tu has been measured. Suppression of both UGA and UAG is observed. There is no significant suppression of any of six UAA sites tested. Nonsense suppression does not require that both genes for EF-Tu be mutant. Strains carrying one mutant and one wild-type tuf gene suppress nonsense mutations. The level of suppression increases approximately additively when both tuf genes are mutant. It is suggested that these mutant forms of EF-Tu act independently of each other to suppress nonsense mutations. Suppression is not observed at all UGA and UAG sites, but instead shows a strong site specificity.     

Transfer of plasmid-borne tuf mutations to the chromosome as a genetic tool for studying the functioning of EF-TuA and EF-TuB in the E. coli cell

The elongation factor EF-Tu of E. coli is a multifunctional protein that lends itself extremely well to studies concerning structure-function relationships. It is encoded by two genes: tufA and tufB. Mutant species of EF-Tu have been obtained by various genetic manipulations, including site- and segment-directed mutagenesis of tuf genes on a vector. The presence of multiple tuf genes in the cell, both chromosomal and plasmid-borne, hampers the characterization of the mutant EF-Tu. We describe a procedure for transferring plasmid-borne tuf gene mutations to the chromosome. Any mutation engineered by genetic manipulation of tuf genes on a vector can be transferred both to the tufA and the tufB position on the chromosome. The procedure facilitated the functional characterization of some of our recently obtained tuf mutations. Of particular relevance is, that it enabled us for the first time to obtain a mutant tufB on the chromosome, encoding an EF-TuB resistant to kirromycin. It thus became possible to study the consequences for growth of tufA inactivation by insertion of bacteriophage Mu. The preliminary evidence obtained suggests that an EF-TuA, active in polypeptide synthesis, is essential for growth whereas such an EF-TuB is dispensable.     

Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement.

The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation. We have constructed fusions between the genes for RF-2 and E. coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable. The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site. The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release). These are the first examples of release factor mutants selectively active for only one of these function. In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis. This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region.     

Divergence among genes encoding the elongation factor Tu of Yersinia Species

Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species.     

Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes. This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon. Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible. A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency. The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone. This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting.     

Suppression of a -1 frameshift mutation by a recessive tRNA suppressor which causes doublet decoding.

sufS was found to suppress the only known suppressible-1 frameshift mutation, trpE91, at a site identified as GGA and mapped within the single gene of the only tRNA that can decode GGA in Escherichia coli. It mapped to the same gene in Salmonella typhimurium. sufS alleles were recessive, and dominant alleles could not be isolated. This is in contrast to all other tRNA structural gene mutations identified thus far that cause frameshift suppression. The recessiveness implies that all sufS alleles are poor competitors against their wild-type tRNA(Gly2) counterparts. The base G immediately 5' of the GGA suppression site influenced the level but was not critical for suppression by sufS601. From this result, it is inferred that sufS601 causes frameshifting by doublet decoding.     

Sequence and functional analysis of mutations in the gene encoding peptide-chain-release factor 2 of Escherichia coli.

Mutations in the prfB gene which encodes peptide-chain-release factor 2 of Escherichia coli were defined by DNA sequence analysis. prfB1 and prfB3 substitute lysine and asparagine for glutamate and aspartate at amino acid positions 89 and 143, respectively. Temperature-sensitive mutations, prfB2 and prfB286, each contain the identical substitution of phenylalanine for leucine-328. These mutations suppress UGA but not UAG or UAA. The efficiency of suppression was affected by the neighboring RNA context. The prfB gene encodes a premature UGA stop codon at position 26 and is expressed by +1 frameshifting. The efficiency of natural frameshift was 18% as measured by using the monolysogenic lambda assay vector containing prfB-lacZ fusions, and increased up to 30% in the prfB mutants. These observations can be interpreted as genetic evidence for the autogenous control of RF2 synthesis by frameshifting. Structural and functional organizations of release factors are discussed.     

Translational frameshifts induced by mutant species of the polypeptide chain elongation factor Tu of Escherichia coli

Translational frameshifts, both +1 and -1, are promoted by mutations in tufA and tufB, the two genes encoding the polypeptide chain elongation factor (EF) Tu of Escherichia coli. Strains harboring the mutant EF-Tu(Ala375----Thr) encoded by either tufA or tufB or by both, display a linear relationship between the frequency of frameshifting and the concentration of mutant EF-Tu, relative to the total amount of EF-Tu. A second mutant species, EF-TuB(Gly222----Asp), also promotes frameshifting. The frequency is strikingly enhanced by the combined action of EF-TuA(Ala375----Thr) and EF-TuB(Gly222----Asp) and exceeds by far the total contribution of the two mutant EF-Tus studied separately. These observations raise the question whether the formation of each peptide bond under conditions that no frameshifting occurs also requires the combined action of two EF-Tu molecules, in this case not differing functionally.     

Use of tRNA suppressors to probe regulation of Escherichia coli release factor 2

It has been suggested that Escherichia coli release factor 2 (RF-2) translation is autoregulated. Mature RF-2 protein can terminate its own nascent synthesis at an intragenic, in-phase UGA codon, or alternatively, a +1 frameshift can occur that leads to completion of the RF-2 polypeptide. Translational termination presumably increases with RF-2 concentration, providing negative regulatory feedback. We now show, in lacZ/RF-2 fusions, that translation of a UAG codon at the position of the UGA competes with frameshifting, which proves one postulate of the translational autoregulatory model. We also identify a nearby sequence that is required for high-frequency frameshifting and suggest a constraint for the codon preceding the shift point. Both these sequences are incorporated into a model for frameshifting. Our measurements allow us to compute the relative rates in vivo of these reactions: release factor action, frameshifting and tRNA selection at an amber codon.     

Evidence for horizontal gene transfer in evolution of elongation factor Tu in enterococci

The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.     

Connection between stop codon reassignment and frequent use of shifty stop frameshifting

Ciliated protozoa of the genus Euplotes have undergone genetic code reassignment, redefining the termination codon UGA to encode cysteine. In addition, Euplotes spp. genes very frequently employ shifty stop frameshifting. Both of these phenomena involve noncanonical events at a termination codon, suggesting they might have a common cause. We recently demonstrated that Euplotes octocarinatus peptide release factor eRF1 ignores UGA termination codons while continuing to recognize UAA and UAG. Here we show that both the Tetrahymena thermophila and E. octocarinatus eRF1 factors allow efficient frameshifting at all three termination codons, suggesting that UGA redefinition also impaired UAA/UAG recognition. Mutations of the Euplotes factor restoring a phylogenetically conserved motif in eRF1 (TASNIKS) reduced programmed frameshifting at all three termination codons. Mutation of another conserved residue, Cys124, strongly reduces frameshifting at UGA while actually increasing frameshifting at UAA/UAG. We will discuss these results in light of recent biochemical characterization of these mutations.     

Is the in-frame termination signal of the Escherichia coli release factor-2 frameshift site weakened by a particularly poor context?

The synthesis of release factor-2 (RF-2) in bacteria is regulated by a high efficiency +1 frameshifting event at an in-frame UGA stop codon. The stop codon does not specify the termination of synthesis efficiently because of several upstream stimulators for frameshifting. This study focusses on whether the particular context of the stop codon within the frameshift site of the Escherichia coli RF-2 mRNA contributes to the poor efficiency of termination. The context of UGA in this recoding site is rare at natural termination sites in E.coli genes. We have evaluated how the three nucleotides downstream from the stop codon (+4, +5 and +6 positions) in the native UGACUA sequence affect the competitiveness of the termination codon against the frameshifting event. Changing the C in the +4 position and, separately, the A in the +6 position significantly increase the termination signal strength at the frameshift site, whereas the nucleotide in the +5 position had little influence. The efficiency of particular termination signals as a function of the +4 or +6 nucleotides correlates with how often they occur at natural termination sites in E.coli; strong signals occur more frequently and weak signals are less common.     

Both genes for EF-Tu in Salmonella typhimurium are individually dispensable for growth

Each of the two genes encoding EF-Tu in Salmonella typhimurium has been inactivated using a mini-Mu MudJ insertion. Eleven independently isolated insertions are described, six in tufA and five in tufB. Transduction analysis shows that the inserted MudJ is 100% linked to the appropriate tuf gene. A mutant strain with electrophoretically distinguishable EF-TuA and EF-TuB was used to show, on two-dimensional gels, that the MudJ insertions result in the loss of the appropriate EF-Tu protein. Southern blotting, using cloned Escherichia coli tuf sequences as probes, shows that each MudJ insertion results in the physical breakage of the appropriate tuf gene. The degree of growth-rate impairment associated with each tuf inactivation is independent of which tuf gene is inactivated. The viability of S. typhimurium strains with either tuf gene inactive contrasts strongly with data suggesting that in the closely related bacterium E. coli, an active tufA gene is essential for growth. Finally the strains described here facilitate the analysis of phenotypes associated with individual mutant or wild-type Tus both in vivo and in vitro.     

The role of EF-Tu and other translation components in determining translocation step size

The two EF-Tu encoding genes, tufA and tufB, of Salmonella typhimurium have been sequenced. Nearly all the differences from their Escherichia coli counterparts are third position changes which do not alter the encoded amino acids. Unexpectedly, most of the changes in one Salmonella tuf gene are paralleled by changes in the other tuf gene perhaps due to gene repair despite the distance separating the genes. Three mutants which cause mis-framing, have their substitutions at codon 375. Explanations for mutants which cause mis-framing are considered and the mechanism of normal reading frame maintenance discussed.     

Mutant ribosomes can generate dominant kirromycin resistance.

Mutations in the two genes for EF-Tu in Salmonella typhimurium and Escherichia coli, tufA and tufB, can confer resistance to the antibiotic kirromycin. Kirromycin resistance is a recessive phenotype expressed when both tuf genes are mutant. We describe a new kirromycin-resistant phenotype dominant to the effect of wild-type EF-Tu. Strains carrying a single kirromycin-resistant tuf mutation and an error-restrictive, streptomycin-resistant rpsL mutation are resistant to high levels of kirromycin, even when the other tuf gene is wild type. This phenotype is dependent on error-restrictive mutations and is not expressed with nonrestrictive streptomycin-resistant mutations. Kirromycin resistance is also expressed at a low level in the absence of any mutant EF-Tu. These novel phenotypes exist as a result of differences in the interactions of mutant and wild-type EF-Tu with the mutant ribosomes. The restrictive ribosomes have a relatively poor interaction with wild-type EF-Tu and are thus more easily saturated with mutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomes are inherently kirromycin resistant and support a significantly faster EF-Tu cycle time in the presence of the antibiotic than do wild-type ribosomes. A second phenotype associated with combinations of rpsL and error-prone tuf mutations is a reduction in the level of resistance to streptomycin.     

A chimeric disposition of the elongation factor genes in Rickettsia prowazekii.

An exceptional disposition of the elongation factor genes is observed in Rickettsia prowazekii, in which there is only one tuf gene, which is distant from the lone fus gene. In contrast, the closely related bacterium Agrobacterium tumefaciens has the normal bacterial arrangement of two tuf genes, of which one is tightly linked to the fus gene. Analysis of the flanking sequences of the single tuf gene in R. prowazekii shows that it is preceded by two of the four tRNA genes located in the 5' region of the Escherichia coli tufB gene and that it is followed by rpsJ as well as associated ribosomal protein genes, which in E. coli are located downstream of the tufA gene. The fus gene is located within the str operon and is followed by one tRNA gene as well as by the genes secE and nusG, which are located in the 3' region of tufB in E. coli. This atypical disposition of genes suggests that intrachromosomal recombination between duplicated tuf genes has contributed to the evolution of the unique genomic architecture of R. prowazekii.     

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 and 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.     

Frameshift suppression through inactivation of translation termination in yeast Saccharomyces cerevisiae: significance of the local context

Site-directed mutagenesis and nucleotide sequence analysis were used to study the roles of the global and local contexts in suppression of the lys2-90 frameshift (FS) mutation in Saccharomyces cerevisiae. Global context features established for the LYS2 mRNA region containing the extra T (lys2-90) were similar to those characteristic of regions involved in translational frameshifting. These were a potential ability of the region to form a pseudoknot and the presence of heptanucleotide CUU UGA C with the "hungry" UGA nonsense codon in the pseudoknot. Some local context features proved to be essential for the phenotypic expression of FS suppression as a result of translational frameshifting. Two amino acid substitutions determined by the nucleotide sequence between the extra U and the UGA nonsense codon lacked expression. A dependence was observed between the efficiency of FS suppression and the type of the nonsense codon located at a particular position downstream of the extra nucleotide (UGA > UAG > UAA). When translation termination was inactivated, nonsense suppression and FS suppression correlated with each other. These results suggest that translational frameshifting, which underlies suppression in the case of inactivation of translation termination, most likely takes place on the nonsense codon arising as a result of insertion of an extra nucleotide.