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.     

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

The structural features of Escherichia coli ribosomal protein S8 that are involved in translational regulation of spc operon expression and, therefore, in its interaction with RNA have been investigated by use of a genetic approach. The rpsH gene, which encodes protein S8, was first inserted into an expression vector under the control of the lac promoter and subsequently mutagenized with methoxylamine or nitrous acid. A screening procedure based on the regulatory role of S8 was used to identify mutants that were potentially defective in their ability to associate with spc operon mRNA and, by inference, 16S mRNA. In this way, we isolated 39 variants of the S8 gene containing alterations at 34 different sites, including 37 that led to single amino acid substitutions and 2 that generated premature termination codons. As the mutations were distributed throughout the polypeptide chain, our results indicate that amino acid residues important for the structural integrity of the RNA-binding domain are not localized to a single segment. Nonetheless, the majority were located within three short sequences at the N terminus, middle, and C terminus that are phylogenetically conserved among all known eubacterial and chloroplast versions of this protein. We conclude that these sites encompass the main structural determinants required for the interaction of protein S8 with RNA.     

Autogenous control is not sufficient to ensure steady-state growth rate-dependent regulation of the S10 ribosomal protein operon of Escherichia coli.

The regulation of the S10 ribosomal protein operon of Escherichia coli was studied by using a lambda prophage containing the beginning of the S10 operon (including the promoter, leader, and first one and one-half structural genes) fused to lacZ. The synthesis of the lacZ fusion protein encoded by the phage showed the expected inhibition during oversynthesis of ribosomal protein L4, the autogenous regulatory protein of the S10 operon. Moreover, the fusion gene responded to a nutritional shift-up in the same way that genuine ribosomal protein genes did. However, the gene did not exhibit the expected growth rate-dependent regulation during steady-state growth. Thus, the genetic information carried on the prophage is sufficient for L4-mediated autogenous control and a normal nutritional shift-up response but is not sufficient for steady-state growth rate-dependent control. These results suggest that, at least for the 11-gene S10 ribosomal protein operon, additional regulatory processes are required to coordinate the synthesis of ribosomal proteins with cell growth rate and, furthermore, that sequences downstream of the proximal one and one-half genes of the operon are involved in this control.     

Characterization of Escherichia coli 50S ribosomal protein L31

The published C-terminal sequence of Escherichia coli 50S ribosomal protein L31, ellipsisRFNK (Brosius, J. (1978) Biochemistry 17, 501-508), differs from that predicted by the gene sequence, ellipsisRFNKRFNIPGSK (GenBank accession no. X78541). This discrepancy might be due to post-translational processing of the protein. To examine this possibility, we have isolated L31 from E. coli strain MRE600 and sequenced the C-terminal tryptic peptide. We find the sequence to be FBIPGSK. Size comparisons of L31 from several E. coli strains demonstrate that all are identical in size to the protein isolated from MRE600 and larger than the previously described protein, indicating that ellipsisRFNKRFNIPGSK represents the true C-terminus of L31. In addition, we show that the failure to identify L31 in many ribosome preparations is probably due to the protein's loose association with the ribosome and its ability to form various intramolecular disulfide bonds, leading to L31 forms with distinct mobilities in gels.     

Mutational alterations of translational coupling in the L11 ribosomal protein operon of Escherichia coli.

The L11 operon in Escherichia coli consists of the genes coding for ribosomal proteins L11 and L1. It is known that translation of L1 does not take place unless the preceding L11 cistron is translated, that is, the two cistrons are translationally coupled, and this is the basis of coregulation of the translation of the two cistrons by a single repressor, L1. Several mutational analyses were carried out to define the region responsible for coupling L1 translation with L11 translation. First, by introducing several amber mutations into the L11 gene by a site-directed mutagenesis technique, it was shown that translation by ribosomes down to a position 21 nucleotides upstream, but not to a position 45 nucleotides upstream, from the end of the L11 cistron allowed the initiation of L11 translation. Second, deletion analysis indicated that a region located 23 to 20 nucleotides from the end of the L11 gene was involved in preventing independent initiation from L1 translation. Third, five different mutations obtained by screening for activation of the masked L1 initiation site were found to be clustered in a small region immediately upstream from the Shine-Dalgarno sequence of L1, and all of them were G-to-A transitions. These results, together with some additional experiments with oligonucleotide-directed mutagenesis, defined the region involved in the coupling and suggest that some special feature of this region, probably different from simple masking of the initiation site by base pairing, is responsible for translational coupling. The present results also suggest that there might be specific differences in the primary nucleotide sequence that distinguish independent translational initiation sites from translationally coupled (i.e., masked) initiation sites.     

Physical characteristics of ribosomal protein S4 from Escherichia coli

A hydrodynamic study of protein S4 from Escherichia coli 30 S ribosomal subunits indicates that this protein is moderately asymmetric. A sedimentation coefficient of 1.69 S and a diffusion coefficient of 7.58 X 10(-7) cm2/s suggest that S4 has an axial ratio of about 5:1 using a prolate ellipsoidal model. This structure should give a radius of gyration of about 29-30 A from small-angle neutron or small-angle x-ray scattering studies. This study has utilized quasi-elastic light scattering as an analytical tool to obtain a diffusion coefficient as well as a method to monitor sample quality. Using quasi-elastic light scattering in this manner allows an assessment of problems associated with protein purity which may be responsible for the many disparate results reported for ribosomal proteins and especially protein S4.     

Escherichia coli ribosomal protein L10 is rapidly degraded when synthesized in excess of ribosomal protein L7/L12.

In Escherichia coli the genes encoding ribosomal proteins L10 and L7/12, rplJ and rplL, respectively, are cotranscribed and subject to translational coupling. Synthesis of both proteins is coordinately regulated at the translational level by binding of L10 or a complex of L10 and L7/L12 to a single target in the mRNA leader region upstream of rplJ. Unexpectedly, small deletions that inactivated the ribosome-binding site of the rplL gene carried on multicopy plasmids exerted a negative effect on expression of the upstream rplJ gene. This effect could be overcome by overproduction of L7/L12 in trans from another plasmid. This apparent stimulation resulted from stabilization of the overproduced L10 protein by L7/L12, presumably because free L10, in contrast to L10 complexed with L7/L12, is subject to rapid proteolytic decay. The contribution of this decay mechanism to the regulation of the rplJL operon is evaluated.     

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.     

Feedback regulation of the rplJL-rpoBC ribosomal protein operon of Escherichia coli requires a region of mRNA secondary structure

The rplJ-rpoBC (L10) operon of Escherichia coli is regulated in part through translational repression (feedback regulation) by ribosomal protein L10 or a complex of ribosomal proteins L10 and L7/L12 (L10-L7/L12). We have constructed mutants in the untranslated leader region of a rplJ-lacZ fusion by oligonucleotide-directed mutagenesis. The mutations include several deletions and a number of single base changes, all of which fail to exhibit normal feedback regulation. Chemical probing of part of the rplJ mRNA leader in the mutagenized region confirms that all of the mutations lie in a stem structure located 140 nucleotides upstream from the translation start-site. The structure includes a 12 base-pair stem, a four base stem-loop, and a six base bulge-loop. Point mutations that abolish feedback regulation are presumed to disrupt this stem structure. Pseudorevertants of selected point mutations were constructed by combining pairs of single base mutations. In these cases, both the secondary structure of the RNA and feedback regulation were restored. The results allow us to define a region of secondary structure in the rplJ mRNA leader that is necessary for feedback regulation.     

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.