A regulatory element within a gene of a ribosomal protein operon of Escherichia coli negatively controls expression by decreasing the translational efficiency

Summary The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kDa protein (21K) of unknown function, the tRNA(m¹G37)methyltransferase (TrmD), and r-protein L19, in this order. Previously we have shown that the steady-state amount of the two r-proteins exceeds that of the 21K and TrmD proteins 12- and 40-fold, respectively, and that this differential expression is solely explained by translational regulation. Here we have constructed translational gene fusions of the trmD operon and lacZ. The expression of a lacZ fusion containing the first 18 codons of the 21K protein gene is 15-fold higher than the expression of fusions containing 49 or 72 codons of the gene. This suggests that sequences between the 18th and the 49th codon may act as a negative element controlling the expression of the 21K protein gene. Evidence is presented which demonstrates that this regulation is achieved by reducing the efficiency of translation.     

Secondary structure of the autoregulatory mRNA binding site of ribosomal protein L1

Summary The secondary structure of the autoregulatory mRNA binding site of Escherichia coli ribosomal protein L1 has been studies using enzymatic methods. The control region of the E. coli L11 operon was cloned into a vector under control of the Salmonella phage SP6 promoter, and RNA transcribed using SP6 RNA polymerase. The secondary structure of this RNA was probed using structure-specific nucleases, and by comparison of the data with computer predictions of RNA folding, secondary structural features were deduced. The proposed model is consistent with elements of some previously proposed models, but differs in other features. Finally, secondary structure information was obtained from two mutant mRNAs and the structural features correlated with observed phenotypes of the mutants.Abbreviations MB mung bean nuclease - V₁ cobra venom nuclease - sss single-strand-specific - dss double-strand-specific     

Genetic studies of the ribosomal proteins in Escherichia coli. 8. Mapping of ribosomal protein components by intergeneric mating experiments between Serratia marcescens and Escherichia coli

Summary Ribosomal protein compositions of Serratia marcescens and Escherichia coli K12 were analyzed by using carboxymethyl cellulose column chromatography. Nine 50S and nine 30S ribosomal proteins of E. coli K12 could be distinguished from those of S. marcescens on the chromatogram.Episomes of E. coli K12, which cover the streptomycin(str) region of the chromosome, were transferred to S. marcescens. Chromatographic analyses were made on the ribosomal proteins extracted from these hybrid strains. At least nine 30S and six 50S ribosomal proteins of E. coli-type could be detected in the ribosomes of the hybrid strains in addition to the ribosomal proteins of S. marcescens.     

The secY-rpmJ region of the spc ribosomal protein operon in Escherichia coli: structural alterations affecting secY expression

Summary A unique feature of the spc ribosomal protein operon is that its region distal to the promoter contains a gene (secY) for an integral membrane protein, followed by an open reading frame termed X which has recently been proposed to encode a new ribosomal protein (protein B). We now show that the open reading frame X indeed directs the synthesis of a protein with electrophoretic mobilities similar to the B protein, and this supports the proposal that X may be more appropriately called rpmJ. Insertion of a plasmid sequence into the secY-rpmJ boundary of the chromosome caused a reduced expression of secY probably by destabilizing the secY part of the message. The results of complementation experiments suggested that a normal level of expression of rpmJ is not required for growth or protein secretion.     

Translational regulation of the L11 ribosomal protein operon of Escherichia coli: mutations that define the target site for repression by L1.

The L11 ribosomal protein operon of Escherichia coli contains the genes for L11 and L1 and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the Shine-Dalgarno sequence for the first cistron, rp1K (L11). By use of a random mutagenesis procedure we have isolated and characterized a series of point mutations in the L11 leader mRNA which eliminate or greatly diminish the regulation by L1. The mutations define a region essential for translational regulation upstream of the L11 Shine-Dalgarno sequence and identify a region of structural homology with the L1 binding site on 23S rRNA. These results are also consistent with the previously proposed model for the secondary structure of the L11 leader mRNA.     

Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ)

Summary The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln⁺ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), an indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.     

DNA sequence of the ribosomal protein genes rp12 and rps19 from a plant-pathogenic mycoplasma-like organism

A segment of a ribosomal protein operon from a plant-pathogenic mycoplasma-like organism (MLO) was cloned and sequenced, to provide supplemental molecular data pertinent to the question of MLO phylogeny. Comparisons of the deduced amino acid sequences indicate an ancient divergence of the MLOs from the animal-pathogenic mycoplasmas. Furthermore, although both the plant and animal pathogens have A-T rich genomes, a fundamental difference was apparent in their usage of the UGA codon.     

A competition mechanism regulates the translation of the Escherichia coli operon encoding ribosomal proteins L35 and L20

Escherichia coli ribosomal protein (r-protein) L20 is essential for the assembly of the 50S ribosomal subunit and is also a translational regulator of its own rpmI-rplT operon, encoding r-proteins L35 and L20 in that order. L20 directly represses the translation of the first cistron and, through translational coupling, that of its own gene. The translational operator of the operon is 450 nt in length and includes a long-range pseudoknot interaction between two RNA sequences separated by 280 nt. L20 has the potential to bind both to this pseudoknot and to an irregular hairpin, although only one site is occupied at a time during regulation. This work shows that the rpmI-rplT operon is regulated by competition between L20 and the ribosome for binding to mRNA in vitro and in vivo. Detailed studies on the regulatory mechanisms of r-protein synthesis have only been performed on the rpsO gene, regulated by r-protein S15, and on the alpha operon, regulated by S4. Both are thought to be controlled by a trapping mechanism, whereby the 30S ribosomal subunit, the mRNA, and the initiator tRNA are blocked as a nonfunctional preternary complex. This alternative mode of regulation of the rpmI-rplT operon raises the possibility that control is kinetically and not thermodynamically limited in this case. We show that the pseudoknot, which is known to be essential for L20 binding and regulation, also enhances 30S binding to mRNA as if this structure is specifically recognised by the ribosome.     

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: structure of the protein and its gene

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.     

Cloning and molecular characterization of the gene rimL which encodes an enzyme acetylating ribosomal protein L12 of Escherichia coli K12

Summary TherimL gene ofEscherichia coli K12 encodes en enzyme catalyzing the acetylation of the N-terminal serine of ribosomal protein L12, thereby converting it into L7. Using a mutant strain defective in this acetylation reaction, we cloned therimL gene into cosmid pHC79 and characterized it at the molecular level. From analysis by SDS-polyacrylamide gel electrophoresis of the proteins synthesized in maxi-cells containing derivatives of therimL-harboring plasmid into which transposon λδ had been inserted at various sites, the product of this gene was identified as a protein with an apparent molecular weight of 20.3 kDa. The nucleotide sequence of the gene and the amino acid sequence deduced from the nucleotide sequence were compared with those of two other ribosomal protein acetylses encoded by therimI andrimJ genes (Yoshikawa et al. 1987). A considerable degree of overall similarity was seen betweenrimL andrimJ, but the degree of similarity betweenrimL andrimI was very low. In addition, a short stretch of similar amino acid sequence was found in all threerim acetylases. The significance of these results with respect to other acetylating enzymes, in particular those involved in the acetylation of aminoglycoside antibiotics is discussed.     

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli

Summary Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M_r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.     

The phenotypic suppression of a mutation in the gene rplX for ribosomal protein L24 by mutations affecting the lon gene product for protease LA in Escherichia coli K12

Summary A suppressor mutation of a temperature-sensitive mutant of ribosomal protein L24 (rplX19) was mapped close to the lon gene by genetic analysis and was shown to affect protease LA. The degradation and the synthesis rates of individual ribosomal proteins were determined. Proteins L24, L14, L15 and L27 were found to be degraded faster in the original rplX19 mutant than in the rplX19 mutant containing the suppressor mutation. Other ribosomal proteins were either weakly or not at all degraded in both mutants. Temperature-sensitive growth was also suppressed by the overproduction of mutant protein L24 from a plasmid. Our results suggest that (1) either free ribosomal proteins or proteins bound to abortive assembly precursors are highly susceptible to the lon gene product and (2) the mutationally altered protein L24 can still function at the nonpermissive growth temperature of the mutant, if it is present in sufficient amounts.     

Structural comparison of 26S rRNA-binding ribosomal protein L25 from two different yeast strains and the equivalent proteins from three eubacteria and two chloroplasts

Summary The sequences ofSaccharomyces carlsbergensis ribosomal protein (r-protein) SL25^* and its equivalents fromCandida utilis (CL25),Escherichia coli (EL23),Bacillus stearothermophilus (BL23),Mycoplasma capricolum (ML23),Marchantia polymorpha chloroplasts (McpL23), andNicotiana tabacum chloroplasts (NcpL23) were examined using a computer program that evaluates the extent of sequence similarity by calculating correlation coefficients for each pair of residues in two proteins from a number of physical properties of individual amino acids. Comparison matrices demonstrate that the prokaryotic sequences (including McpL23 and NcpL23) can be aligned unambiguously by introducing small internal deletions/insertions at three specific positions. A similar comparison brought to light a clear evolutionary relationship between the prokaryotic and the yeast proteins despite the fact that visual inspection of these sequences revealed only limited similarity. The alignment deduced from this comparison shows the two yeast r-proteins to have acquired a long (50–60 amino acids) N-terminal extension as well as a 13-amino acid-long deletion near the C-terminus. The significance of these findings in terms of the evolution of r-proteins in general and the biological function of various parts of the SL25 protein in particular is discussed.     

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: Structure of the protein and its gene

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.     

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.     

The nucleotide sequence of an Escherichia coli operon containing genes for the tRNA(m1G)methyltransferase, the ribosomal proteins S16 and L19 and a 21-K polypeptide.

The nucleotide sequence of a 4.6-kb SalI-EcoRI DNA fragment including the trmD operon, located at min 56 on the Escherichia coli K-12 chromosome, has been determined. The trmD operon encodes four polypeptides: ribosomal protein S16 (rpsP), 21-K polypeptide (unknown function), tRNA-(m1G)methyltransferase (trmD) and ribosomal protein L19 (rplS), in that order. In addition, the 4.6-kb DNA fragment encodes a 48-K and a 16-K polypeptide of unknown functions which are not part of the trmD operon. The mol. wt. of tRNA(m1G)methyltransferase determined from the DNA sequence is 28 424. The probable locations of promoter and terminator of the trmD operon are suggested. The translational start of the trmD gene was deduced from the known NH2-terminal amino acid sequence of the purified enzyme. The intercistronic regions in the operon vary from 9 to 40 nucleotides, supporting the earlier conclusion that the four genes are co-transcribed, starting at the major promoter in front of the rpsP gene. Since it is known that ribosomal proteins are present at 8000 molecules/genome and the tRNA-(m1G)methyltransferase at only approximately 80 molecules/genome in a glucose minimal culture, some powerful regulatory device must exist in this operon to maintain this non-coordinate expression. The codon usage of the two ribosomal protein genes is similar to that of other ribosomal protein genes, i.e., high preference for the most abundant tRNA isoaccepting species. The trmD gene has a codon usage typical for a protein made in low amount in accordance with the low number of tRNA-(m1G)methyltransferase molecules found in the cell.