Cooperative effects by the initiation codon and its flanking regions on translation initiation.

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo.

The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.

Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli.

The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.

Interaction between a mutant release factor one and P-site peptidyl-tRNA is influenced by the identity of the two bases downstream of the stop codon UAG.

Termination efficiency of a mutant form of RF (release facor) 1, as compared to the wild-type enzyme, is influenced by the P-site peptidyl-tRNA if the termination signal is UAGA. This effect is weaker at the stronger termination signal UAGU. Similarly, low efficiency of the mutant RF1, together with certain peptidyl-tRNAs, can be increased by changing the second base of the 3'-flanking codon from C to G. The data suggest that the mutant RF1 interacts with the P-site peptidyl-tRNA in conjunction with the context at the 3'-side of the termination codon.

The influence of 5' codon context on translation termination in Saccharomyces cerevisiae.

Translation termination in vivo was studied in the yeast Saccharomyces cerevisiae using a translation-assay system. Codon changes that were made at position -2 relative to the stop codon, gave a 3.5-fold effect on termination in a release-factor-defective (sup45) mutant strain, in line with the effect observed in a wild-type strain. The influence of the -2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient termination. Thus, the C-terminal end of the nascent peptide influences translation termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing - 1 (P-site) codons in yeast gave a 10-fold difference in effect on the efficiency of termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso-codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in termination efficiency. This suggests that the P-site tRNA is able to influence termination at UGAC in yeast.

The influence of the 5' codon context on translation termination in Bacillus subtilis and Escherichia coli is similar but different from Salmonella typhimurium.

The last two amino acids in the nascent peptide influence translation termination in E. coli (Mottagui-Tabar et al., 1994; Björnsson et al., 1996). We have compared the effects on termination in Escherichia coli, Bacillus subtilis and Salmonella typhimurium obtained by varying the -1 and -2 codons upstream of the weak UGAA stop signal. The peptide effect from the penultimate amino acid on translation termination in B. subtilis is similar to that seen in E. coli (with 66.5% RF-2 amino acid sequence similarity), whereas the influence in S. typhimurium (with 95.3% similarity to E. coli) is weaker. The effect of changing the -1 codon (P-site) is weaker in S. typhimurium as compared to those in E. coli and B. subtilis. RF-2s from E. coli and S. typhimurium have a threonine or alanine at position 246, respectively. This amino acid exchange in RF-2 can explain the difference in efficiency and toxicity during overexpression when E. coli and S. typhimurium are compared (Uno et al., 1996). However, B. subtilis RF-2 also has an alanine at that position, yet the sensitivity to the nascent peptide is similar to that in E. coli. Thus, the amino acid difference at position 246 in the RF-2 sequences cannot explain why termination in E. coli and B. subtilis is similar in peptide sensitivity while being different from that in S. typhimurium. Sequence alignments of RF-2 from the three bacteria show other regions of the molecule that could be involved in the functional interactions with the C-terminal end of the nascent peptide.

Visualization of a large conformation change of ribosomes in Escherichia coli cells starved for tryptophan or treated with kirromycin.

Computer-aided electron tomography has been used to visualize ribosomes in Escherichia coli cells treated with kirromycin. This antibiotic stops bacterial growth by blocking the release of EF-Tu. GDP from the ribosome after GTP cleavage. Ribosomes in the kirromycin-treated cells are very compact, with the two subunits in close contact with each other. This closed structure is different from the open structure with spatially separated subunits that characterizes ribosomes in tryptophan-starved cells, giving deficiency for tryptophanyl.tRNA. A comparison of ribosomes in exponentially growing bacteria suggests that most ribosomes in an undefined working mode are in the closed conformation.

Physiologically dependent appearance of a low density region that corresponds to the tunnel through the 50S part of the 70S ribosome.

Ribosomes have different conformations in cells that are starved for a required amino acid (giving aminoacyl.tRNA starvation), or treated with kirromycin (blocking EF-Tu.GDP release), or are in exponential growth. A tunnel spans the 50S ribosome from a location facing the 70S ribosomal intersubunit space to the back side of the subunit in Escherichia coli cells. Here we have analyzed the internal low density region that corresponds to this tunnel in ribosomes in vivo. The data suggest that the tunnel is opened in connection with spatial separation of the subunits in ribosomes that have an empty A-site due to starvation for aminoacyl.tRNA. A region that corresponds to this tunnel can be found in the more compact structure of ribosomes in kirromycin-treated cells only after a substantial removal of low density material. This region is even less prominent in ribosomes in undefined working modes in growing bacteria. The data suggest that appearance of the tunnel through the 50S ribosomal subunit is working-mode dependent and it is not a characteristic feature of the major fraction of the ribosomal population in growing cells.

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Growth phase dependent stop codon readthrough and shift of translation reading frame in Escherichia coli.

Nonsense codon readthrough and changed translational reading frame were measured in different growth phases in E. coli. The strains used carry plasmid constructs with a translation assay reporter gene. This reporter gene contains an internal stop codon or a run of U-residues. Termination or frameshifting give rise to stable proteins that can be physically quantified on gels along with the complete protein products. Readthrough of the stop codon UGA by a nearcognate tRNA is several fold higher in active growth than in late exponential phase. In early exponential phase, about 7% of -1 frameshift at a U9 slippery sequence is detectable; upon entry to stationary phase this frameshifting increases to about 40% followed by a decrease in stationary phase. A similar increase is observed in the case of +1 reading frameshift at the U9 sequence, which increases from 13% in early exponential growth phase up to 38% at the beginning of stationary phase followed by a decrease. Thus, the levels of both stop codon readthrough and frameshifting are growth phase dependent, though not in an identical fashion.

Functional interaction between tRNA2Gly2 at the ribosomal P-site and RF1 during termination at UAG.

The glycine codons GGA or GGG, on the 5' side of stop codons UAG and UGA, are associated with a uniquely low termination efficiency in Escherichia coli, as compared to other codons, including the two glycine codons GGU and GGC. In contrast to the wild-type strain, all four glycine codons have a similar effect on termination at UAG in a strain with a mutant release factor 1 (RF1). Thus, these two glycine codon pairs, when present at the ribosomal P-site, affect termination efficiency of mutant or wild-type RF1 at UAG differently. If reading of GGA/G by tRNAGly2 is eliminated in the RF1 wild-type strain and replaced by a mutant form of tRNAGly3, termination efficiency is increased to the same level as for GGU/C, normally read by tRNAGly3. The results suggest an unusual interaction between the P-site tRNAGly2 and wild-type RF1 at the ribosomal A-site that is not present with mutant RF1.

The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo.

A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.

Only the last amino acids in the nascent peptide influence translation termination in Escherichia coli genes.

Efficiency of translation termination is affected if the last two amino acids in the nascent peptide are changed [1,2]. By changing the corresponding codons upstream of the stop signal UGAA, we have analyzed if the -3 to -6 amino acids at the C-terminal region of the nascent peptide also affect termination. Lysine at position -3 gave increased readthrough, whereas a total of 28 variations at positions -4, -5, and -6 showed no significant effect on readthrough. The 3'-ends corresponding to the last six codons in 27 Escherichia coli genes were inserted upstream of a stop codon in the 3A' translation assay gene [1]. Readthrough of the stop codon was measured and a possible correlation with the Codon Adaptation Index (CAI) 131 of the respective genes was investigated. Sequences from genes with low CAI do not give any such correlation, whereas sequences from genes with high CAI values are correlated with high termination efficiency. This correlation disappears if the -1 and -2 codons/amino acids are changed. The results suggest that mainly the terminal dipeptide of the terminal hexapeptide sequence has an influence on termination in the tested E. coli genes. This influence is dependent on the charge of the -2 amino acid and is correlated with the alpha-helix propensity of the -1 amino acid, in accordance with results obtained from synthetic gene constructs [1,2].

Automated correlation and averaging of three-dimensional reconstructions obtained by electron tomography.

We have developed a least-squares refinement procedure that in an automated way performs three-dimensional alignment and averaging of objects from multiple reconstructions. The computer implementation aligns the three-dimensional structures by a two-step procedure that maximizes the density overlap for all objects. First, an initial average density is built by successive incorporation of individual objects, after a global search for their optimal three-dimensional orientations. Second, the initial average is subsequently refined by excluding individual objects one at a time, realigning them with the reduced average containing all other objects and including them into the average again. The refinement is repeated until no further change of the average occurs. The resulting average model is therefore minimally biased by the order in which the individual reconstructions are incorporated into the average. The performance of the procedure was tested using a synthetic data set of randomly oriented objects with Poisson-distributed noise added. The program managed well to align and average the objects at the signal/noise ratio 1.0. The increase in signal/noise ratio was in all investigated cases almost equal to the expected square root of the number of objects. The program was also successfully tested on a set of authentic three-dimensional reconstructions from an in situ specimen containing Escherichia coli 70S ribosomes, where the immediate environment of the reconstructed objects may also contain variable amounts of other structures.

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

Functional interaction between release factor one and P-site peptidyl-tRNA on the ribosome.

Translation termination at UAG is influenced by the nature of the 5' flanking codon in Escherichia coli. Readthrough of the stop codon is always higher in a strain with mutant (prfA1) as compared to wild-type (prfA+) release factor one (RF1). Isocodons, which differ in the last base and are decoded by the same tRNA species, affect termination at UAG differently in strains with mutant or wild-type RF1. No general preference of the last codon base to favour readthrough or termination can be found. The data suggest that RF1 is sensitive to the nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site). For some isoaccepting P-site tRNAs (tRNA3(Pro) versus tRNA2(Pro), tRNA4(Thr) versus tRNA1,3Thr) the effect is different on mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself. The glycine codons GGA (tRNA2(Gly)) and GGG (tRNA2,3(Gly)) at the ribosomal P-site are associated with an almost threefold higher readthrough of UAG than any of the other 42 codons tested, including the glycine codons GGU/C, in a strain with wild-type RF1. This differential response to the glycine codons is lost in the strain with the mutant form of RF1 since readthrough is increased to a similar high level for all four glycine codons. High alpha-helix propensity of the last amino acid residue at the C-terminal end of the nascent peptide is correlated with an increased termination at UAG. The effect is stronger on mutant compared to wild-type RF1. The data suggest that RF1-mediated termination at UAG is sensitive to the nature of the codon-anticodon interaction of the wobble base, the last amino acid residue of the nascent peptide chain, and the tRNA at the ribosomal P-site.

Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon.

A RNA fragment which is protected from degradation by ribosome pausing at a stop codon has been identified in growing Escherichia coli. The fragment is 261 nt long and corresponds to the 3'-end of the mRNA expressed from a semi-synthetic model gene. The 5'-end of the RNA fragment, denoted rpRNA (ribosomal pause RNA), is located 13 bases upstream of the stop codon. In vivo decay of the complete mRNA and accumulation of rpRNA are dependent on the nature of the stop codon and its codon context. The data indicate that the rpRNA fragment arises from interrupted decay of the S3A'mRNA in the 5'-->m3'direction, in connection with a ribosomal pause at the stop codon. RF-2 decoding of UGA is less efficient than RF-1 decoding of UAG in identical codon contexts, as judged from rpRNA steady-state levels. The half-life of UGA-containing rpRNAs is at least 5 min, indicating that ribosomal pausing can be a major factor in stabilising downstream regions of messenger RNAs.

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.