Translational regulation of the L11 ribosomal protein operon of Escherichia coli: analysis of the mRNA target site using oligonucleotide-directed mutagenesis.

Abstract

The Lii ribosomal protein operon in Escherichia coli consists of the genes for proteins Lii and Li and is feedback regulated by the translational repressor L1. The mRNA target site for this repression is located close to the translation initiation site of the first Lii cistron. Several mutant plasmid molecules carrying altered nucleotide sequences in the Li target site were constructed by site-directed in vitro mutagenesis using synthetic oligodeoxyribonucleotides. Specifically, we examined the importance of a presumptive double-stranded stem structure that is common among Li binding sites on rRNA from a variety of organisms and in LI1 mRNA. Mutational alterations that disrupt the stem structure were found to abolish translational regulation as analyzed both in vitro and in vivo. Two of the mutations were combined so that the stem structure was restored but with a different primary nucleotide sequence. This double mutant was shown to restore the original phenotype, the ability to be translationally regulated by L1. These experiments show the importance of the stem structure, but not its primary sequence, for the interaction of Li with the mRNA and support the concept that mRNA target sites share some structural features with the corresponding ribosomal protein binding sites of rRNA. MATERIALS AND METHODS Oligodeoxyribonucleotides used for mutagenesis were prepared by the phosphotriester synthesis method (10). dT-T-A-T-T-A-C-G-G-A-A-C-T-T-G-A-G was synthesized in thislaboratory and purified by polyacrylamide gel electrophoresis. Other oligonucleotides (see Fig. 3) were synthesized by Bruce Kaplan (City of Hope Research Institute) and were purified by HPLC. Mutagenesis directed with these oligonucleotides was carried out essentially as described by Zoller and Smith (1i). DNA sequence determination of mutant plasmids was carried out by the method of Maxam-Gilbert-.(12) from the HindIII site in the EcoRI fragment used as the target DNA for mutagenesis (see Fig. 2). Standard techniques were used for construction of various plasmids and M13 phage derivatives (13,14).DNA-dependent in vitro protein synthesis was carried out as described (2, 15). Protein Li, purified previously (16), was stored at -70'C and used for the in vitro experiments as described (2,6). Measurements of relative synthesis rates of r proteins in vivo were carried out as described in the legend to 5389The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

“…This sequestration, in turn, may provide the rpsF operon with a regulatory feedback mechanism that ensures the balanced and coordinated synthesis of the proteins encoded. In analogous systems, associated with other r-protein operons, the regulatory effect is either entirely coupled (e.g., affects equally translation of all genes) or weakens in downstream genes (Nomura et al 1980;Baughman and Nomura 1984). As most of the bacterial rpsF operons encode both components of the S6:S18 complex, the coupled mechanism of regulation appears to be more appealing.…”

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

“…la). pNO2625 and pN02731 are very similar to the Ai-6 (pN01587) and A2 deletion plasmids, respectively, used in previous studies (2,3). The construction of pNO2625 is schematically shown in Fig.…”

“…The 3H/14C ratios were then determined for each r-protein as described previously (3), and the 3H/14C ratio for each r-protein was normalized to the 3H/'4C ratio of total protein in the sample. The values obtained in this way represent differential synthesis rates of each r-protein relative to those in i4C-labeled reference cells and are given in the tables as relative differential synthesis rates.…”

“…First, we assumed that translation of Lii from pNO2625 plasmid mRNA and from chromosomal mRNA was equally sensitive to Li repression and that the rate of transcription of the Lll-Li mRNA increased 10-fold after induction of the plasmid operon (2,3). Thus, in the strain carrying pNO2625 with the MB6 mutation, 1/10 of the total Lii synthesis is from the chromosomal operon (i.e., 0.03) and the remaining 9/10 is from the plasmid operon (0.27).…”

“…An example is the regulation of the Lii operon, which consists of the genes for Lii (rplK) and Li (rplA). It was demonstrated that one of the gene products, the Li protein, acts as a repressor to inhibit the synthesis of both Lii and Li at the level of translation (13,43) by acting at a single target site which is near the translation initiation site for Lii (2,3,44). The inhibition of Lii translation by Li takes place directly, and that of Li translation takes place only indirectly as a result of the inhibition of Lii translation; the translation of Li does not take place unless the preceding Lii cistron is translated, that is, the two cistrons are translationally coupled.…”