Translation through an uncDC mRNA secondary structure governs the level of uncC expression in Escherichia coli

Abstract

Escherichia coli expresses the and E subunits of F1F0-ATP synthase at relative levels consistent with the 3:1 (,B/£) stoichiometry in the holoenzyme. The mechanism of translational control of expression of the uncC gene (£ subunit) relative to the immediately 5' uncD gene (,1 subunit) was examined. Previous expression studies and a computer analysis suggested the presence of an RNA secondary structure including the 3' end of uncD, the uncDC intergenic region, and the uncC Shine-Dalgarno sequence (S. D. Dunn and H. G. Dallmann, J. Bacteriol. 172:2782Bacteriol. 172: -2784Bacteriol. 172: , 1990 expressed from the same transcript, control of their relative levels of expression must be exerted at the posttranscriptional level. Translational control of protein biosynthesis is a method widely used for regulation of gene expression in bacteria (for recent reviews, see references 7, 20, and 33) and, in general, is exerted at the level of translation initiation rather than elongation (21, 23). For genes located in polycistronic operons, features of the translation initiation regions (TIRs) including the intrinsic sequence of the region as well as potential for mRNA secondary-structure formation largely govern the efficiency of translation (10,20,41,45).The potential for mRNA to form secondary structures provides a powerful method of control of gene expression for genes contained in polycistronic messages. Secondary structures which can sequester critical regions of a TIR limit the accessibility of the translation machinery to an mRNA and reduce expression (7,42 We have previously shown that an inhibitory effect of upstream uncD sequences on the expression of the uncC gene was correlated with a predicted mRNA secondary structure involving the uncC TIR and 72 bases at the 3' end of the uncD coding region (12). In the present paper, we report the physical determination of the secondary structure of in vitro-transcribed uncDC RNA and demonstrate that -subunit synthesis is partially coupled to translation of the uncD gene. MATERIALS AND METHODSBacterial strains and growth. The E. coli strains used in this work are listed in Table 1. Strain JM103 was used in the construction of all plasmids and was grown in Luria-Bertani medium at 37°C with shaking.Strain AN1460 was the source of purified Fj-ATPase used as a standard and was grown as described elsewhere (13). Strains AN724 and AN1008 were used in 13 and -expression studies and were grown in 2 x YT medium (31) at 37°C with shaking. When required, these cultures were supplemented with arginine, uracil, and 2,3-dihydroxybenzoic acid each to 40 ,ug/ml.

The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated

“…This activation Involves the destabilization of an inhibitory secondary structure in the mRNA (Mayford and Weisblum, 1985} which allows a second ribosome to initiate at the downstream Shine-Dalgarno sequence. In some cases the ribosome-binding site {RBS) of the translationally regulated gene is actually sequestered within the inhibitory secondary structure (Dallman and Dunn, 1994). However, in other situations (as appears to be the case for pilK) the RBS is not sequestered in a RNAiRNA duplex but is located close enough to the stem of the secondary structure to inhibit ribosomal access (Blumer et aL, 1987).…”

“…The efficiency of coupling or the degree to which the downstream gene (i.e., pilK) is expressed, therefore, depends upon the length of time for which the 'activated' sequences remain available for translation initiation (Blumer et al. 1987;Dallman and Dunn, 1994). In theory, it should be possible to estimate the duration of p/7K translation initiation region availability and the approximate frequency with which pilK is translated.…”

Mechanism of translational coupling in the nifLA operon of Klebsiella pneumoniae

“…By this mechanism, ribosomes translating the proximal gene would melt the inhibitory mRNA secondary structure, thus allowing new ribosomes to gain access from solution to the RBS of the distal gene. Facilitated binding has been suggested for other translationally coupled genes (Lesage et al ., 1992; Dallmann and Dunn, 1994), and it may be the only mechanism to explain the synthesis of the distal gene product in greater amounts than the proximal one, as is observed in some coupled systems (Yates and Nomura, 1981; Gerstel and McCarthy, 1989). However, unambiguous direct evidence regarding the involvement of the same or different ribosomes in the translation of a pair of translationally coupled genes has not yet been provided.…”

Gonococcal Genes Encoding Transferrin-Binding Proteins A and B Are Arranged in a Bicistronic Operon but Are Subject to Differential Expression

“…The presence of an inverted repeat between tbpA and tbpB is unique to this gene cluster; neither the genes that encode the components of the lactoferrin receptor (lbpBA) nor those that encode the hemoglobin receptor (hpuAB) are separated by similar sequence or spacing (4,5,35,36,46). The stoichiometry of multisubunit complexes, such as the photosynthesis reaction center (33) and the bacterial ATPase (16), are influenced by secondary structures located in the polycistronic operons from which they are encoded. Furthermore, the stem-loop structures in these systems influence the relative amounts of the subunit transcripts and thereby affect the final stoichiometry of the complex.…”