Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli

Expression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome-binding site. By measuring the effect of mutations on the expression of a translational rpsO-lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.     

Translational autocontrol of the Escherichia coli ribosomal protein S15

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.     

Stability of ribosomal protein mRNA and translational feedback regulation in Escherichia coli

Summary It was previously observed that the stability of ribosomal protein (r-protein) mRNA in Escherichia coli decreases under the conditions where its translation is feedback inhibited by repressor r-protein. We have now demonstrated that the stability of mRNA for r-proteins S13, S11 and S4 increases in a strain carrying a mutation in the gene for S4, a translational repressor regulating these r-proteins. The results confirm the previous observations that translational repression increases the decay rate of r-protein mRNA, and in addition, show that the half-life of S13-S4 r-protein mRNA in cells growing under ordinary conditions is significantly shorter than its inherent stability would predict, due to the operation of translational feedback regulation.     

Structural elements of rps0 mRNA involved in the modulation of translational initiation and regulation of E. coli ribosomal protein S15.

Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.     

The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.     

Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli.

We have purified a small subunit mitochondrial ribosomal protein, MRPS28p, from the yeast, Saccharomyces cerevisiae. Sequence from the amino terminus of MRPS28p was used to design a degenerate oligonucleotide that was complementary to the MRPS28 gene. The MRPS28 gene was isolated and its sequence determined. The MRPS28 sequence encodes a 28 kDa protein that has a region of homology with ribosomal protein S15 of E. coli. This region spans the entire length of the E. coli protein, but as MRPS28p is larger, includes only the portion of the MRPS28p sequence from amino acids 150 to 238. Based on this homology, we predict that MRPS28p, like E. coli S15, interacts directly with small subunit rRNA and functions as an early protein in ribosome assembly. Cells carrying a disrupted chromosomal copy of MRPS28 are unable to respire and spontaneously lose portions of their mitochondrial genomes at a high frequency. These phenotypes are consistent with an essential role for MRPS28p in the assembly and/or function of the mitochondrial ribosome.     

Cloning of rpsO, the gene for ribosomal protein S15 of Escherichia coli

Summary The gene for Escherichia coli ribosomal protein S15 (rpsO) was cloned on the vector pBR322 from F-prime JCH55 DNA. The recombinant plasmid was transformed to Serratia marcescens cells and it was proved that E. coli S15 was synthesized and incorporated into ribosome particles in S. marcescens cells. A DNA fragment containing rpsO was also inserted into the vector pRF3, which changes its copy number depending on the growth temperature in a temperature-sensitive polA host. By use of this recombinant plasmid it was shown that the relative synthesis rate of S15 increased about twice even when the copy number of the plasmid increased more than twenty-fold.     

Influence of translational efficiency on the stability of the mRNA for ribosomal protein S20 in Escherichia coli.

A set of plasmids was constructed so as to contain point mutations which limit the efficiency and/or extent of translation of the gene for ribosomal protein S20. These plasmids were transformed into strains carrying mutations in the genes for polynucleotide phosphorylase (pnp-7), RNase E (rne-1), or both. Subsequently, the effect of translational efficiency on mRNA abundance and chemical half-life was determined. The data indicated that mutations altering translational efficiency also affected mRNA levels over a 10-fold range. This variation in mRNA abundance occurred independently of mutations in either RNase E or polynucleotide phosphorylase, both of which determine the stability of the S20 mRNAs. Moreover, a mutation at codon 15 which caused premature termination of translation of the S20 mRNA did not significantly reduce its stability in different genetic backgrounds. We propose a model in which initiation of translation competes for early steps in mRNA turnover, which could be the binding of RNase E itself or as a complex to one or more sites near the 5' terminus of the S20 mRNA.     

Structure of the Escherichia coli S10 ribosomal protein operon.

The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data.     

Post-translational modification of Escherichia coli ribosomal protein S6.

Summary Escherichia coli has multiple forms of ribosomal protein S6, differing in number of glutamyl residues at the C-terminal end. Three forms are revealed when crude cell extracts are fractionated by a two-dimensional gel electrophoresis technique. Pulse-chase experiments show that the shortes and most alkaline form of S6 is the first to appear. In about one doubling time this form reaches equilibrium with the two other forms of S6, implicating the existance of an enzyme, which adds glutamic acid residues to S6. We show that the relative levels of these three S6 forms are not affected by the growth rate of the culture.     

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

A long-range RNA-RNA interaction forms a pseudoknot required for translational control of the IF3-L35-L20 ribosomal protein operon in Escherichia coli.

In the IF3-L35-L20 operon encoding translation initiation factor 3 (IF3) and the two ribosomal proteins L35 and L20, the expression of the genes that code for the two ribosomal proteins is negatively regulated at the translational level by the cellular concentration of L20. This translational repressor directly regulates the expression of the gene encoding L35 and, via translational coupling, that of its own gene. Mutations that affect the control of the L35 gene were found exclusively at two sites: the first is located approximately 300 nucleotides upstream, and the second immediately 5' of the translation initiation site of the L35 gene. Mutations that fall between these two sites have little or no effect on the control, and the lack of effect of a deletion in the intervening region confirms this finding. RNA structure mapping in vitro suggests that the first site pairs with the second. We show that this pairing is also likely to occur in vivo because single mutations in either of these sites affect control, but base pair compensatory mutations re-establish control. We propose that these two distant sites can base-pair to form a long-range pseudoknot which is required for the control of the expression of the L35 gene.     

Isoaspartate in ribosomal protein S11 of Escherichia coli

Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its beta-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal alpha-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins in E. coli differed dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.