Spc Operon Research Articles

The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene.

The genes encoding the 52 ribosomal proteins (r-proteins) of Escherichia coli are organized into approximately 19 operons scattered throughout the chromosome. One of these, the spc operon, contains the genes for ten ribosomal proteins: L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15 (rp1N, rp1X, rp1E, rpsN, rpsH, rp1F, rp1R, rpsE, rpmD, and rp1O). We now report the entire 5.9 kb nucleotide sequence of the spc operon. DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon. It has also revealed the presence of two open reading frames past the last known gene (L15) of the spc operon. One of these corresponds to a gene (pr1A or secY) which recently has been shown by others to be involved in protein export. In addition, S1 mapping experiments indicate that a significant proportion of transcription initiated from the spc operon continues not only into the two putative genes, but also without termination into the downstream alpha r-protein operon.

A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery

The gene prlA codes for a factor that appears to function in the export of proteins in Escherichia coli. This conclusion is based on the finding that mutations altering the priA gene product restore export of envelope proteins with defective signal sequences. Previous results showed that the prlA gene lies in an operon (spc) known to code for ten different ribosomal proteins. Our studies show that the prlA gene lies promoter-distal to the last known ribosomal protein gene in this operon. Evidence from gene fusions constructed in vitro suggests that prlA codes for a protein containing at least 300 amino acids. Thus a heretofore unidentified protein specified by a gene within the spc operon appears to be a component of the cellular protein export machinery.

Expression of Ribosomal Genes in Bacteria

Publisher Summary This chapter discusses the molecular genetics of ribosomal proteins (r-proteins) and ribosomal ribonucleic acid (rRNA) in E. coli. Several molecular mechanisms for regulating r-protein and rRNA synthesis include autogenous regulation of r-protein operons by key regulatory r-proteins encoded by the operon, which they regulate; differential regulation of the transcription of the β operon by attenuation at a site between two genes in the operon; and regulation of the transcription of ribosomal genes by modulating the promoter specificity of RNA polymerase. The capacity for total protein synthesis in fast-growing cells is determined by the number, rather than the activity, of the ribosomes. Only a fraction of the ribosomes in more slowly growing cells are active in protein synthesis, but the active ribosomes translate at the same rate as found in the fast-growing cells. A significant characteristic of the r-protein gene expression in diploid strains was revealed by the measurements of r-protein messenger RNA (mRNA) synthesis. The synthesis of mRNA from the spc operon is increased two-fold compared to the messenger from “control” operons in a strain lysogenic for a phage carrying the spc operon. Thus, the r-protein synthesis can be regulated at the posttranscriptional level.

Interconnection between assembly and synthesis of ribosomal proteins.

The effect of a defect in ribosome assembly on the rate of synthesis of ribosomal proteins was studied with three cold-sensitive mutants of Escherichia coli which possess mutations in the structural genes for ribosomal proteins S5 (rpsE), S8 (rpsH) or L6 (rplF8). The rate of protein synthesis was determined at different times after the shift to the nonpermissive condition. The following results were obtained: (i) The blocking of assembly induces a distinct polarity in the expression of most of the ribosomal protein transcriptional units. In the spc operon, this polarity affects the expression of genes “downstream” from each of the respective mutant alleles. (ii) Despite the fact that the formation of ribosomal proteins is greatly unbalanced at low temperature there is no accumulation of most of the ribosomal proteins (except L5 and L25) in the cytosol. This cannot be explained on the basis of autoregulation since the expression of the structural genes of some autoregulatory ribosomal proteins (e.g. S4) is much lower than that of non-regulating (but coregulated) proteins (e.g. S13). (iii) The polar effect reduces the expression of genes of ribosomal proteins from both subunits and may thereby introduce an interdependence of 30S and 50S synthesis. In the S5 mutant, the 5S rRNA binding proteins L5 and L25 are accumulated in the cytoplasm under the condition of unbalanced assembly whereas L18 is incorporated. This can be interpreted in the way that the reduced synthesis of L-proteins (L15 or L30) prevents L5 and L25 from incorporation into 50S precursor. (iv) Protein S20 is found bound both to 30S and 50S particles under nonpermissive condition and may participate both in 30S and 50S assembly.

Translational regulation by ribosomal protein S8 in Escherichia coli: structural homology between rRNA binding site and feedback target on mRNA.

It has been previously shown that ribosomal protein synthesis in Escherichia coli is regulated at the level of translation by certain key ribosomal proteins. In the spc operon, S8 regulates the expression of L5 and some of the subsequent genes, while the first two genes (L14 and L24) are regulated independently. We therefore determined the DNA sequence at the junction of the L24 and L5 genes, which corresponds to the putative feedback target for S8. We show that there is a striking homology between the structure of the mRNA for this region and the known binding site for S8 on 16S rRNA. These results support the theory that the regulation of ribosomal protein synthesis is based on competition between rRNA and mRNA for regulatory ribosomal proteins.

Escherichia coli ribosomal protein S8 feedback regulates part of spc operon.

In Escherichia coli the genes coding for the 52 ribosomal proteins (r-proteins) are organized into a number of transcription units located at various regions on the bacterial genome. The expression of r-protein genes is balanced so that individual r-protein synthesis rates change coordinately in response to changing environmental conditions, and significant amounts of free r-proteins do not exist in the cellular pool. We have suggested a model for the balanced regulation of r-protein gene expression, namely that r-protein synthesis and ribosome assembly are coupled so that r-proteins not incorporated into ribosomes prevent the further translation of r-protein mRNA by a feedback regulatory mechanism. The model was tested in vitro by examining the effect of purified r-proteins on DNA directed r-protein synthesis, and in vivo by examining the effect of overproduction of certain r-proteins on the synthesis rates of other r-proteins. In vitro experiments have revealed that some r-proteins (L1, L4, L10, S4 and S8) can selectively inhibit the synthesis of r-proteins whose genes are in the same operon as their own, and that this specific feedback regulation occurs at the level of translation rather than at the level of transcription of mRNA. Regulatory roles for L1, S4 and L4 have also been established by in vivo experiments. We have studied further the feedback regulatory properties of S8 in vivo and in vitro, and report here that the protein regulates a part of the spc operon.

Isolation of E. coli mutants containing multiple transpositions of IS sequences.

The characterization of three E. coli mutants that appeared to have unselected IS1 insertions on the chromosome are described. One had a single new IS1 sequence. The second had three new IS1 sequences. The third had two new IS1 sequences and one of the IS1 sequences in the parent was missing. These mutants were found in a collection on strains that contained IS insertions in the spc operon. The frequency of finding mutants with unselected IS1 transpositions was at least 100 times greater than expected. The results suggest several transposition events may frequently occur in the same cell.

In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins.

Isolation and characterization of stable hybrid mRNA molecules transcribed from ribosomal protein promoters in E. coli

The promoters from the str and spc operons of ribosomal proteins from E. coli were inserted into the Hind II cleavage site of mini-Col E1 (pVH51) plasmid. For both promoters, strains with the hybrid plasmid accumulated a small RNA species not present in strains carrying the vector. These RNAs were analyzed by RNA sequencing techniques and compared to DNA sequences. In both cases, synthesis of the new RNA species is initiated by the cloned r protein promoter at the site predicted by previous in vitro experiments. The RNAs extend across the Hind II site used for cloning and terminate specifically in the vector sequences. The termination site was localized to six consecutive thymine nucleotides preceded by a sequence with dyad symmetry. We found that the RNA from the str promoter was 205 (± 3) nucleotides long and that from the spc promoter was 177 (±3) nucleotides long. These “hybrid mRNAs” are much more stable than ordinary mRNA. The str hybrid mRNA has a half-life of about 8 min, and the spc hybrid mRNA has a half-life of about 18 min at 37°C. These hybrid mRNAs provide an in vivo system with which to examine directly the discrete transcription products from ribosomal protein promoters, and to study promoter function and mRNA metabolism in vivo.

Regulation of ribosomal protein synthesis in Escherichia coli by selective mRNA inactivation.

In an Escherichia coli strain lysogenic for lambda spc2 transducing phage, an extra copy of ribosomal protein (r-protein) genes in the spc and alpha operons are carried on the phage chromosome. Expression of genes in the spc operon in this merodiploid strain was compared with that in a control "haploid" strain carrying lambda trkA phage. It was found that the synthesis rate of spc mRNA, relative to other reference mRNA in the merodiploid strain, is about 2-fold higher than that in the control strain; yet, no dosage effect was observed in the synthesis rate of r-proteins in the spc or alpha operon. The spc mRNA was found to be more rapidly degraded in the merodiploid strain than in the control strain, and its steady-state amount, relative to reference mRNA, was only slightly higher in the merodiploid strain than in the control strain. Thus, E. coli cells have the ability to regulate the rate of r-protein synthesis regardless of the rate of transcription of r-protein genes, presumably by inactivation of the mRNA followed by its degradation. A model is proposed which involves selective inactivation of r-protein mRNA by a feedback mechanism. The model can explain coordinated synthesis of r-proteins and other observations related to selective expression of certain alleles in diploid strains.

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.

DNA sequences of promoter regions for the str and spc ribosomal protein operons in E. coli

The DNA sequences have been determined for promoter regions of two ribosomal protein operons in E. coli, the str operon and the spc operon. The site of in vitro transcription initiation within each of these promoter regions has been determined. The start site of the str operon occurs 69 bases upstream from the initiation codon of the S12 gene. The start site of the spc operon occurs 72 bases upstream from the L14 gene, and only 91 bases downstream from the termination codon of the S17 gene (which is in the preceding S10 operon). Both promoters are similar to other sequenced promoters in that they each have an identifiable “Pribnow box” sequence 5 bases upstream from the transcription start site. The spc promoter has a long sequence of 2 fold symmetry centered within the Pribnow box; the str promoter has a shorter but similar symmetry. At positions −69 through −40 in the spc operon, another long region of symmetry is present which may be the termination signal of the preceding S10 operon. Extensive sequence similarity between the str and spc promoter regions is found downstream from the Pribnow box-that is, in a transcribed region preceding the translation start sites.