Adenosylcobalamin inhibits ribosome binding to btuB RNA

Abstract

Expression of the btuB gene encoding the outer membrane cobalamin transporter in Escherichia coli is strongly reduced on growth with cobalamins. Previous studies have shown that this regulation occurs in response to adenosylcobalamin (Ado-Cbl) and operates primarily at the translational level. Changes in the level and stability of btuB RNA are consequences of the modulated translation initiation. To examine how Ado-Cbl affects translation, the binding of E. coli 30S ribosomal subunits to btuB RNA was investigated by using a primer extension inhibition assay. Ribosome binding to btuB RNA was much less efficient than to other RNAs and was preferentially lost when the ribosomes were subjected to a high-salt wash. Ribosome binding to btuB RNA was inhibited by Ado-Cbl but not by cyanocobalamin, with half-maximal inhibition around 0.3 microM Ado-Cbl. Ribosome-binding activity was increased or decreased by mutations in the btuB leader region, which affected two predicted RNA hairpins and altered expression of btuB-lacZ reporters. Finally, the presence of Ado-Cbl elicited formation of a single primer extension-inhibition product with the same specificity and Cbl-concentration dependence as the inhibition of ribosome binding. These results indicate that btuB expression is controlled by the specific binding of Ado-Cbl to btuB RNA, which then affects access to its ribosome-binding sequence.

Figure 1

(A Upper) Schematic structure of the btuB regulatory region. The positions of regulatory elements are indicated relative to the transcription start site and the start of the btuB coding sequence. The region from +1 to +315 is carried on the DNA template for synthesis of btuB RNA, with transcription from the T7 φ10 late promoter; the region for primer hybridization in the toe-print assay is indicated (Right). (Lower) The major primer extension products are identified by the nucleotide position of their 3′ end. (B Lower) Ribosome binding to lac and btuB RNA. RNA containing the translation initiation region from lacZ and btuB genes were synthesized by using T7 RNA polymerase and used in the primer extension inhibition assay. Incubation in the presence of tRNA^fMet, 20 nM 30S ribosomal subunits, or 5 μM Ado-Cbl, as indicated (Upper), was for 10 min; primer extension was then carried out for 10 min after addition of AMV reverse transcriptase. The four lanes (Left) (A,C,G,T) are sequencing ladders for the btuB region; (Right) the positions of the last base of the primer extension fragments.

Figure 2

Dependence of lac and btuB RNA binding on concentration of 30S ribosomal subunits. Ribosome binding is measured as the relative amount of the T256 product, expressed as a percentage of the major primer extension products. Binding of unwashed (open symbols) and high-salt-washed (filled symbols) ribosomes to lac (circles) and btuB (squares) RNA. Binding of unwashed ribosomes to btuB RNA in the presence of 5 μM Ado-Cbl is indicated (triangles). Curves are fit to a hyperbolic binding process.

Figure 3

Effect of cobalamins on the amount of btuB primer extension inhibition products. Binding reactions were carried out in the presence of the indicated concentrations of Ado-Cbl (circles), methyl-Cbl (squares), and CN-Cbl (diamonds), with 200 nM 30S subunits and 1 μM tRNA^FMet. (A) Ribosome binding to the translation initiation site to generate fragment T256. (B) Formation of cobalamin-dependent A202 product. (C) Data from A (open symbols) and B (closed symbols) are plotted as the percent of the maximal effect to compare the effect of Ado-Cbl (circles) and methyl-Cbl (squares) on inhibition of ribosome binding (open symbols) and formation of fragment A202 (closed symbols) vs. cobalamin concentration.

Figure 4

Predicted secondary structure of btuB RNA in the region from nucleotides 171 to 243. The sequence changes of mutants described in this study are indicated, along with the positions of hairpins 1 and 2. The start codon is boxed and the position of the 3′ end of the A202 product is indicated by the arrow.

Figure 5

Ribosome binding to variant btuB RNAs. Primer extension assay was carried out with RNAs carrying the indicated mutations (described in Fig. 4). Ribosomal 30S subunits were present at 200 nM, and 5 μM Ado-Cbl was present as indicated. The identity of the primer extension products are indicated on the sides of the figure.