L11 Operon Research Articles

Characterization of Regulatory Elements of L11 and L1 Operons in Thermophilic Bacteria and Archaea.

Ribosomal protein L1 is a conserved two-domain protein that is involved in formation of the L1 stalk of the large ribosomal subunit. When there are no free binding sites available on the ribosomal 23S RNA, the protein binds to the specific site on the mRNA of its own operon (L11 operon in bacteria and L1 operon in archaea) preventing translation. Here we show that the regulatory properties of the r-protein L1 and its domain I are conserved in the thermophilic bacteria Thermus and Thermotoga and in the halophilic archaeon Haloarcula marismortui. At the same time the revealed features of the operon regulation in thermophilic bacteria suggest presence of two regulatory regions.

Identification of Ribosomal Protein L1-Binding Sites in Thermus thermophilus and Thermotoga maritima mRNAs

The conserved two-domain ribosomal protein (r-protein) L1 is a structural part of the L1 stalk of the large ribosomal subunit and regulates the translation of the operon that comprises its own gene. The regulatory properties of the bacterial r-protein L1 have only been studied in detail for Escherichia coli; however, there were no such studies for other bacteria, in particular, Thermus thermophilus and Thermotoga maritima, which are more evolutionarily ancient. It is known that domain I of the r-protein L1 might have regulatory properties of the whole protein. The aim of this study was to identify regulatory sites on the mRNA of T. thermophilus and T. maritima that interact with r-proteins L1, as well as with their domains I from the same organisms. An analysis of the mRNA of the L11 operon T. thermophilus showed the presence of one potential binding site of the L1 r-protein, two such regions were found also in the mRNA sequence of the L11 operon of T. maritima. The dissociation constants for the L1 proteins from T. thermophilus and T. maritima and their domains I with mRNA fragments from the same organisms that contain the supposed L1-binding sites were determined by surface plasmon resonance. It has been shown that the ribosomal proteins L1 as their domains I bind specific fragments of mRNA from the same organisms that may suggest regulatory activity of the L1 protein in the T. thermophilus and T. maritima and conservatism of the principles of L1-RNA interactions.

MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own translation before or at the formation of the first peptide bond.

The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail. As in Escherichia coil, regulation takes place at the level of translation. MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E. coli, was shown to be an autoregulator of the MvaL1 operon. The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome. Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1. Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies. In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron. A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co-regulatory function of MvaL10, the homologue of the regulatory protein L10 of the beta-operon of E. coil. However, we show that MvaL10 does not have a regulatory function.

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.

Mutational analysis of the L1 binding site of 23S rRNA in Escherichia coli.

The L11 ribosomal protein operon of Escherichia coli contains the genes for L11 and L1 and is feedback regulated by the translational repressor L1. Both the L1 binding site on 23S rRNA and the L1 repressor target site on L11 operon mRNA share similar proposed secondary structures and contain some primary sequence identity. Several site-directed mutations in the binding region of 23S rRNA were constructed and their effects on binding were examined. For in vitro analysis, a filter binding method was used. For in vivo analysis, a conditional expression system was used to overproduce a 23S rRNA fragment containing the L1 binding region, which leads to specific derepression of the synthesis of L11 and L1. Changes in the shared region of the 23S rRNA L1 binding site produced effects on L1 binding similar to those found previously in analysis of corresponding changes in the L11 operon mRNA target site. The results support the hypothesis that r-protein L1 interacts with both 23S rRNA and L11 operon mRNA by recognizing similar features on both RNAs.

Cloning and DNA sequence determination of the L11 ribosomal protein operon of Serratia marcescens and Proteus vulgaris: translational feedback regulation of the Escherichia coli L11 operon by heterologous L1 proteins.

In Escherichia coli the genes encoding ribosomal proteins L11 (rplK) and L1 (rplA) are contained in a single operon and their expression is translationally regulated by L1. We have cloned the homologous genes from two other enterobacteria, Serratia marcescens and Proteus vulgaris, and determined nucleotide sequences. The genes are organized in a similar way to that found in E. coli. Conservation of nucleotide and amino acid sequences relative to E. coli in the protein coding regions are 89.2% and 94.7% for S. marcescens, and 80.9% and 88.6% for P. vulgaris. Nucleotide sequences of L11 mRNA leader regions were strongly conserved for the primary as well as the secondary structures in the L1 target site. We have also constructed plasmids carrying E. coli L11 and either P. vulgaris or S. marcescens L1 genes fused to the lac promoter, with or without the E. coli leader containing the L1 target site. Induction of transcription of the operons possessing the E. coli mRNA leader did not lead to overproduction of L11, indicating translational regulation of the chimeric operon as well as the chromosomal operon by the plasmid encoded L1. Repression of the chromosomal L11 operon was directly demonstrated upon induction of the chimeric operons without the leader, which also lack the L11 initiation signal but have a mutation allowing L1 translation.(ABSTRACT TRUNCATED AT 250 WORDS)

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

The secondary structure of the autoregulatory mRNA binding site of Escherichia coli ribosomal protein L1 has been studied 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.

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.

Translational regulation is responsible for growth-rate-dependent and stringent control of the synthesis of ribosomal proteins L11 and L1 in Escherichia coli.

The physiological importance of translational regulation in controlling the synthesis of ribosomal proteins from the L11 ribosomal protein operon was determined for the classical regulatory phenomena of growth rate dependence and stringent control. Translational regulation of the L11 operon by ribosomal protein L1, the L11 operon-specific translational repressor protein, was abolished by introducing a chromosomal mutation that causes an alteration of the site where L1 interacts with L11 operon mRNA. It was found that abolishing translational regulation of the L11 operon also abolished growth-rate-dependent regulation and stringent control of the L11 operon ribosomal proteins without affecting the normal regulation of ribosomal proteins from other operons that are not regulated by L1. These results show that both growth-rate-dependent control and stringent control of ribosomal protein synthesis in the L11 operon are a direct result of translational regulation.

Changes in the half-life of ribosomal protein messenger RNA caused by translational repression

The half-life of ribosomal protein operon L11 mRNA in vivo was measured during exponential growth by following the kinetics of incorporation of radioactive precursors into L11 mRNA transcribed from multi-copy plasmids. The degree of translational feedback regulation by L1, the L11 operon-specific translational repressor protein, was changed by altering the site on the “L11 mRNA” where L1 interacts. The half-life of the overproduced L11 mRNA increased by about fivefold when translational repression was abolished, while the half-life of mRNA from the spc ribosomal protein operon, which is not translationally regulated by L1, stayed constant. Furthermore, the half-life of L11 operon mRNA carrying an additional mutation in the ribosome binding site that abolishes translation remains short. This indicates that the change in half-life observed during increased gene dosage is due to translational repression by L1 and is probably a consequence of L1 blocking translation of L11 mRNA and not due to some nucleolytic activity mediated by L1.

Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli

The L11 ribosomal protein operon in Escherichia coli consists of the structural genes for proteins L11 and L1. Hybrid deletion plasmids were constructed carrying these two genes with decreasing amounts of the leader mRNA under lac transcriptional control. Measurements of mRNA and protein synthesis directed by these plasmids in vitro and in vivo demonstrate that the regulation of this operon is post-transcriptional and identifies a region of the mRNA, preceding the proximal L11 gene, important for successful feedback inhibition of L11 and L1 synthesis by L1. Additionally, deletions extending to the ribosome binding site of the L11 gene fail to synthesize both L11 and the downstream L1 protein although synthesis of the corresponding mRNA remains unchanged. These results directly demonstrate the presence of translational coupling in this bicistronic operon.

Feedback regulation of ribosomal protein synthesis in E. coli: Localization of the mRNA target sites for repressor action of ribosomal protein L1

E. coli ribosomal protein L1 is a translational repressor of the synthesis in vitro of both proteins encoded in the L11 operon (L11 and L1). L1 is shown to act at a single target site within the first 160 bases of the bicistronic mRNA, near (or at) the translation initiation site of the L11 cistron. Synthesis of L1 apparently requires translation of the preceding L11 cistron, allowing regulation of the synthesis of both proteins from a single mRNA target site. This observation suggests a sequential translation mechanism that results in the equimolar synthesis rates of the two proteins observed in vivo. It was found that the presence of 23S rRNA, but not 16S rRNA, relieves translational inhibition by L1. L1 presumably recognizes structural features of the mRNA target site that are homologous to the L1-binding site of 23S rRNA. Although previous work indicated that translationally inhibited ribosomal protein mRNA is degraded in vivo, L1 repressor action in the present in vitro system was found not to involve mRNA degradation.

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.

Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli.

The 3072-nucleotide-long sequence of a segment from the 88-min region of the Escherichia coli chromosome has been determined. The sequence covers the genes for ribosomal proteins L11 (rplK), LI (rplA), L10 (rplJ), and L7/L12 ((rplL), and the 5' end of the gene for the beta subunit of RNA polymerase (rpoB), along with the presumed regulatory regions for these genes. The probable locations of the promoter for the first two genes (the L11 operon) and the promoter for the latter three genes (the proximal part of the beta operon) have been identified. We have also found that the four ribosomal protein genes preferentially use codons that are recognized efficiently by the most abundant tRNA species. These and other features of the sequence results are discussed in relation to available information obtained from both in vitro and in vivo experiments on the expression of these ribosomal and RNA polymerase subunit genes.