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.     

Autogenous control: ribosomal protein L10-L12 complex binds to the leader sequence of its mRNA

Ribosomal proteins L10 and L12 are encoded in the L10 operon, situated at position 89.5 min on the Escherichia coli genetic map, and are able to regulate their own translation. The two proteins form a L10-L12 complex that is able to bind specifically to the leader sequence of the L10 operon mRNA and prevent translation. We show that the leader sequence: (i) is required for the translation of mRNA into L10 and L12 proteins; and (ii) contains a unique binding site for the inhibitory L10-L12 complex. We suggest that a specific secondary structure of the leader RNA is required for translation. When this structure is perturbed by L10-L12 binding, by deletion, or point mutations, translation is inhibited. The block on the synthesis of L10 and L12 can presumably be removed by the incorporation of the inhibitory L10-L12 complex into assembling 50S ribosome subunits. We observed that rRNA prevents the binding of L10-L12 to the mRNA. Furthermore, we have identified extended sequence homologies within the 23S rRNA and L10 leader region RNA. The L10-L12 binding site on the mRNA includes part of the homologous sequences.     

In vivo and in vitro structural analysis of the rplJ mRNA leader of Escherichia coli. Protection by bound L10-L7/L12

The rplJL-rpoBC operon of Escherichia coli is regulated in part at the level of translation by an autogenous mechanism (feedback regulation) that involves ribosomal protein L10-L7/L12. Feedback regulation occurs as the result of L10-L7/L12 binding to a site on the untranslated leader region of the rplJ mRNA that is located more than 100 nucleotides upstream from the translation start site. Previous studies have indicated that the secondary structure of the rplJ leader region is important for efficient translation and feedback regulation. We have done chemical modification experiments to examine the secondary structure of approximately 200 nucleotides of the rplJ leader region, and we propose a secondary structure that is consistent with the experimental data. RNA structure was probed in vitro by treating samples of total cellular RNA with diethyl pyrocarbonate and in vivo by treating log-phase cultures with dimethyl sulfate. Modified bases were detected by primer extension using three different oligonucleotide primers. The proposed structure includes five double-stranded regions designated I to V, separated by single-stranded segments numbered 1 to 5. We have also identified specific nucleotides in the rplJ mRNA leader that are protected by purified L10-L7/L12 from methylation by dimethyl sulfate in vitro. The protected bases are located within a bulge-loop of region IV, a portion of the mRNA that has been shown genetically to be necessary for feedback regulation.     

Importance of the leader region of mRNA for translation initiation of ColE2 Rep protein

Translation initiation of mRNA encoding the Rep protein of the ColE2 plasmid required for initiation of plasmid DNA replication is fairly efficient in Escherichia coli cells despite the absence of a canonical Shine-Dalgarno sequence. To define sequences and structural elements responsible for translation efficiency of the Rep mRNA, a series of rep-lacZalpha translational fusions bearing various mutations in the region encoding the leader region of the Rep mRNA was generated and tested for the translation activity by measuring the beta-galactosidase activity. We showed that the region rich in A and U between the stem-loop II structure and GA cluster sequence, formation of the stem-loop II structure, but not its sequence, and the region between the GA cluster sequence and initiation codon are important along with the GA cluster sequence for efficient translation of the Rep protein. The existence of these important regions in the leader region of the Rep mRNA may explain the mechanism of inhibition of the Rep protein translation by an antisense RNA (RNAI), which is complementary to the leader region.     

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.     

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.     

Domains of the Escherichia coli threonyl-tRNA synthetase translational operator and their relation to threonine tRNA isoacceptors.

The expression of the gene for threonyl-tRNA synthetase (thrS) is negatively autoregulated at the translational level in Escherichia coli. The synthetase binds to a region of the thrS leader mRNA upstream from the ribosomal binding site inhibiting subsequent translation. The leader mRNA consists of four structural domains. The present work shows that mutations in these four domains affect expression and/or regulation in different ways. Domain 1, the 3' end of the leader, contains the ribosomal binding site, which appears not to be essential for synthetase binding. Mutations in this domain probably affect regulation by changing the competition between the ribosome and the synthetase for binding to the leader. Domain 2, 3' from the ribosomal binding site, is a stem and loop with structural similarities to the tRNA(Thr) anticodon arm. In tRNAs the anticodon loop is seven nucleotides long, mutations that increase or decrease the length of the anticodon-like loop of domain 2 from seven nucleotides abolish control. The nucleotides in the second and third positions of the anticodon-like sequence are essential for recognition and the nucleotide in the wobble position is not, again like tRNA(Thr). The effect of mutations in domain 3 indicate that it acts as an articulation between domains 2 and 4. Domain 4 is a stable arm that has similarities to the acceptor arm of tRNA(Thr) and is shown to be necessary for regulation. Based on this mutational analysis and previous footprinting experiments, it appears that domains 2 and 4, those analogous to tRNA(Thr), are involved in binding the synthetase which inhibits translation probably by interfering with ribosome loading at the nearby translation initiation site.     

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.     

S4-alpha mRNA translation regulation complex. II. Secondary structures of the RNA regulatory site in the presence and absence of S4

The secondary structure of the Escherichia coli alpha mRNA leader sequence has been determined using nucleases specific for single- or double-stranded RNA. Three different length alpha RNA fragments were studied at 0 degrees C and 37 degrees C. A very stable eight base-pair helix forms upstream from the ribosome initiation site, defining a 29 base loop. There is evidence for base-pairing between nucleotides within this loop and for a "pseudo-knot" interaction of some loop bases with nucleotides just 3' to the initiation codon, forming a region of complex structure. A weak helix also pairs sequences near the 5' terminus of the alpha mRNA with bases near the Shine-Dalgarno sequence. Affinity constants for the translational repressor S4 binding different length alpha mRNA fragments indicate that most of the S4 recognition features must be contained within the main helix and hairpin regions. Binding of S4 to the alpha mRNA alters the structure of the 29 base hairpin region, and probably melts the weak pairing between the 5' and 3' termini of the leader. The pseudo-knot structure and the conformational changes associated with it provide a link between the structures of the S4 binding site and the ribosome binding site. The alpha mRNA may therefore play an active role in mediating translational repression.     

A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.