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.     

RNA secondary structure and translation inhibition: analysis of mutants in the rplJ leader

We have carried out measurements of the stable binding of the ribosomal protein (r-protein) complex L10-L7/L12 to mutant forms of the mRNA leader of the rplJ operon of Escherichia coli. One of the point mutations, base 1548, which lies within the L10-L7/L12-protected region, almost completely abolishes in vitro formation of a stable complex of L10-L7/L12 with rplJ mRNA leader, and a second point mutation, base 1634, strongly reduces it. These observations constitute strong support for the proposition that L10-L7/L12 binds to the rplJ leader in bringing about translational feedback. To account for the action of these and other mutations, and to explain the mechanism of translation feedback inhibition, we suggest a secondary structure model involving alternate forms of the rplJ mRNA leader.     

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.     

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.     

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.     

Mutations in the rpIJ leader of Escherichia coli that abolish feedback regulation

We have isolated mutants that fail to exhibit biosynthetic feedback regulation of a rpIJ-lacZ fusion. Analysis of these mutants and of others that were isolated earlier indicates that crucial sequences for both translational feedback regulation and efficient translation lie closely intermingled in the central region of the rpIJ mRNA leader 70-195 bases upstream from the translation start of rpIJ. We suggest that our point mutations define a region of the rpIJ leader mRNA to which L10 binds in effecting autogenous translational regulation.     

Chemical and enzymatic probing of spatial structure of the omega leader of tobacco mosaic virus RNA

The 5′-untranslated sequence of tobacco mosaic virus RNA — the so-called omega leader — exhibits features of a translational enhancer of homologous and heterologous mRNAs. The absence of guanylic residues, the presence of multiple trinucleotide CAA repeats in its central region, and the low predictable probability of the formation of an extensive secondary structure of the Watson-Crick type were reported as the peculiarities of the primary structure of the omega leader. In this work we performed chemical and enzymatic probing of the secondary structure of the omega leader. The isolated RNA comprising omega leader sequence was subjected to partial modifications with dimethyl sulfate and diethyl pyrocarbonate and partial hydrolyses with RNase A and RNase V1. The sites and the intensities of the modifications or the cleavages were detected and measured by the primer extension inhibition technique. The data obtained have demonstrated that RNase A, which attacks internucleotide bonds at the 3′ side of pyrimidine nucleotides, and diethyl pyrocarbonate, which modifies N7 of adenines not involved in stacking interactions, weakly affected the core region of omega leader sequence enriched with CAA-repeats, this directly indicating the existence of a stable spatial structure. The significant stability of the core region structure to RNase A and diethyl pyrocarbonate was accompanied by its complete resistance against RNase V1, which cleaves a polyribonucleotide chain involved in Watson-Crick double helices and generally all A-form RNA helices, thus being an evidence in favor of a non-Watson-Crick structure. The latter was confirmed by the full susceptibility of all adenines and cytosines of the omega polynucleotide chain to dimethyl sulfate, which exclusively modifies N1 of adenines and N3 of cytosines not involved in Watson-Crick interactions. Thus, our data have confirmed that (1) the regular (CAA)_n sequence characteristic of the core region of the omega leader does form stable secondary structure, and (2) the structure formed is not the canonical double helix of the Watson-Crick type.     

Effect of ermC leader region mutations on induced mRNA stability.

Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence. Another result of ribosome stalling is stabilization of ermC mRNA. The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region. Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure. Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability. A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation. This mutation was used to measure the effect of mRNA stabilization on ermC gene expression.     

Structure of the 5'' untranslated regulatory region of ferritin mRNA studied in solution.

Ferritin mRNAs are the first eukaryotic mRNAs for which a conserved, translational regulatory sequence has been identified. The sequence of twenty-eight nucleotides, called the IRE (iron regulatory element), is found in the 5'-noncoding region and is required for enhanced translation of ferritin mRNA by excess cellular iron; regulation occurs at initiation. The prediction of secondary structure in the IRE is a hairpin loop. We now report an analysis of the IRE structure in solution studied in natural ferritin mRNAs [H and H'(M) subunits] by primer extension, after modification or cleavage by dimethyl sulfate, RNAases T1 and V1, and the chemical nuclease 1, 10-phenanthroline-copper (OPCu) which cleaves single-stranded and bulged regions of RNA. Overall, the structure in solution of the ferritin mRNA regulatory region is a hairpin loop, with magnesium-sensitive features, in which half the stem is provided by the IRE and half by flanking regions; only secondary structure is conserved in the flanking regions. Predicted bulges or internal loops along the stem were clearly detected by OPCu but were missed by the more bulky probe RNAase T1, indicating the efficacy of OPCu in probing subtle features of RNA structure. Magnesium-dependent deviations from the predicted structure were observed in the stem between the hairpin loop and the bulge at C6. The location of the IRE in relation to the initiator AUG or the cap is variable in different ferritin mRNAs. However, the number of nucleotides in the base-paired flanking regions of known ferritin mRNAs is proportional to the distance of the IRE from the cap and places the secondary/tertiary structure 8-10 nucleotides from the cap where interference with initiation is likely.     

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.     

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.     

Characterization of the Escherichia coli 23S Ribosomal RNA region associated with ribosomal protein L1. Evidence for homologies with the 5''-end region of the L11 operon.

The results previously obtained upon studying the L1-23S RNA complex by the fingerprint technique have been reexamined in the light of new data on 23S RNA primary structure. The 23S RNA region that remains associated with the L1 ribosomal protein after RNase digestion of the synthetic complex lies between nucleotides 2067 and 2235 from the 5'-end of the molecule. This region contains a m7G near to the 5'-end and possesses a high degree of mutability in E. coli. Three different sequences were observed in E. coli MRE 600. All three sequences differ in two positions relative to the corresponding sequence in rrnB cistron from E. coli K12. Striking homology is observed between the 23S RNA region associated with protein L1 and the 5'-part of L11 operon. This observation supports the model of feedback regulation of r-proteins synthesis proposed by Yates et al. (PNAS, 77, 1837) and strongly suggests that the region of 23S RNA located between positions 2155 and 2202 is essential for the binding of protein L1.