Feedback regulation of the spc operon in Escherichia coli: translational coupling and mRNA processing

Abstract

The spc operon of Escherichia coli encodes 10 ribosomal proteins in the order L14, L24, L5, S14, S8, L6, L18, S5, L30, and L15. This operon is feedback regulated by S8, which binds near the translation start site of L5 and inhibits translation of L5 directly and that of the distal genes indirectly. We constructed plasmids carrying a major portion of the spc operon genes under lac transcriptional control. The plasmids carried a point mutation in the S8 target site which abolished regulation and resulted in overproduction of plasmidencoded ribosomal proteins upon induction. We showed that alteration of the AUG start codon of L5 to UAG decreased the synthesis rates of plasmid-encoded distal proteins, as well as L5, by approximately 20-fold, with a much smaller (if any) effect on mRNA synthesis rates, indicating coupling of the distal cistrons' translation with the translation of L5. This conclusion was also supported by experiments in which S8 was overproduced in trans. In this case, there was a threefold reduction in the synthesis rates of chromosome-encoded L5 and the distal spc operon proteins, but no decrease in the mRNA synthesis rate. These observations also suggest that transcription from ribosomal protein promoters may be special, perhaps able to overcome transcription termination signals. We also analyzed the state of ribosomal protein mRNA after overproduction of S8 in these experiments and found that repression of ribosomal protein synthesis was accompanied by stimulation of processing (and degradation) of spc operon mRNA. The possible role of mRNA degradation in tightening the regulation is discussed.It is known that the synthesis of many of the ribosomal proteins (r-proteins) in Escherichia (0li is coordinately and stoichiometrically balanced with the assembly of mature ribosomes and that' a posttranscriptional feedback mechanism is largely responsible for this regulation (for reviews, see references 17, 21,'and 28). Key r-proteins have been identified which, when synthesized in excess of the need for ribosome assembly, act as translational feedback inhibitors on their polycistronic mRNA to control the synthesis of all r-proteins within their regulatory unit.How does a single repressor r-protein inhibit the synthesis of'more than one r-protein? This question was first studied with the Lii operon, which encodes the genes for Lii and LI and is regulated by Li. It was demonstrated that Li interacts with mRNA at a single target site near the translation start site of Lii and inhibits translation of both Lii and Li (2,43). The inhibition of Lii translation by Li takes place directly and that of Li translation takes place only indirectly as a result of inhibition of Lii synthesis. It was shown that the synthesis of Li does not take place unless the preceding Lii cistron is translated, that is, LI synthesis is translationally coupled to Lii synthesis, and this is the basis of coregulation of the two genes by the single translational repressor LI (2,35,43). Recent experiments have also demonstrated the presen...

Mutagenesis of ribosomal protein S8 from Escherichia coli: defects in regulation of the spc operon

“…Overexpression of wild-type S8 from plasmid-borne copies of the rpsH gene is believed to impede cell growth because the excess S8 binds to, and consequently inhibits the translation of, chromosomally encoded spc operon mRNA (12). Mutant (32). Judging from the immunoprecipitation assay, the amount of S8 in bacteria harboring pZNO28 increased by a factor of 4 to 6 under our conditions.…”

“…When expressed in excess, S8 is thought to bind to a specific site in the polycistronic spc mRNA, inhibiting the translation of at least eight ribosomal proteins and causing a significant reduction in the growth rate (12,32,39). A fragment of the mRNA that is protected from nuclease digestion by S8 was isolated and found to contain almost all of the characteristic primary-and secondary-structure features of the S8 binding site in 16S rRNA, although a slight difference in the affinity of the protein for the two sites ensures its preferential association with the rRNA (19).…”

Cloning and sequence analysis of the Chlamydia trachomatis spc ribosomal protein gene cluster

“…However, the mechanism of transcriptional regulation differs among E. coli r-protein operons (24)(25)(26). Recently, one group reported the regulation of spc operon r-genes by translational coupling in which the product of EcoS8 gene binds to the spc operon mRNA near the beginning of EcoL5, leading to repression of EcoL5 translation and subsequently the downstream spc operon (31). The same group (15), however, failed to observe similar structures in the B. subtilis gene cluster, prompting them to speculate that the translational feedback regulation system for control of r-protein gene expression is fairly recent in evolution.…”

“…Although a few other ribosomal determinants have been identified, r-protein gene organization and expression have not been well characterized (7-10, 20, 21). Interestingly, the organization and transcriptional regulation of r-protein operons appear to be well conserved among eubacteria; however, their regulation among evolutionary distant species remains unelucidated (15).Recently, we reported the cloning and sequencing of the r-protein CtrL6e from C. trachomatis, which is structurally and functionally homologous to Escherichia coli r-protein EcoL6 (13 (6,31). In Bacillus subtilis and Thennus aquaticus, however, no structure resembling an S8 translational repressor has been identified, suggesting some different mechanism of transcriptional regulation (15, 18).…”

“…Recently, we reported the cloning and sequencing of the r-protein CtrL6e from C. trachomatis, which is structurally and functionally homologous to Escherichia coli r-protein EcoL6 (13 (6,31). In Bacillus subtilis and Thennus aquaticus, however, no structure resembling an S8 translational repressor has been identified, suggesting some different mechanism of transcriptional regulation (15, 18).…”