The highly efficient translation initiation region from the Escherichia coli rpsA gene lacks a shine-dalgarno element

Abstract

The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position -91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the "specialized ribosome" approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.

FIG. 1.

(Top) Secondary structure of E. coli rpsA TIR and site-directed mutations introduced in the sequence. The TIR is folded in three successive hairpins (I, II, and III) separated by single-stranded regions (ss1 and ss2), as determined by chemical probing experiments and phylogenetic comparisons (3). The online mfold (version 3.1) computer program (26, 45) predicts energy parameters of −10.8, −11.4, and −2.7 kcal/mol for hairpins I, II, and III, respectively. The sequence is numbered from the first base of the initiator codon; mutated positions are in boldface. (Bottom) For each mutated rpsA TIR used in this work, the combined mutations are listed according to their position in the sequence. A dash indicates a sequence identical to the wild-type (WT) sequence.

FIG. 2.

(A) The genetic system used here. Plasmid and E. coli chromosomes are schematically shown as large rectangles. The plasmid-borne rrnB operon (gray boxes) is under control of the arabinose-inducible P_BAD promoter. Sequences of the anti-SD borne by the pASD^WT and pASD^mut plasmids are indicated. Only one of the seven chromosomal rrn operons is represented (open boxes), along with the IPTG-inducible lacZ chromosomal fusion used as the translational reporter (hatched and closed boxes). (B) Schematic representation of the rpsA TIR, indicating possible base pairings with the wild-type (ASD^WT) or specialized (ASD^mut) ASD. The upper drawing corresponds to the wild-type TIR, whereas in the lower one the GGA motifs in loops I and II are changed to CCU. Additional mutations introduced in each TIR are indicated below the sequence. For clarity, the weak hairpin III is represented unfolded.

FIG. 3.

Expression and translational activity of specialized ribosomes. (A) The plasmid-encoded rRNA, which carries the C1192U mutation, can be distinguished from chromosome-encoded rRNA by primer extension. The relevant 16S rRNA region is illustrated, together with the 17-mer used as a primer (dotted arrow). In the presence of ddGTP (see Materials and Methods), primer extension on chromosome and plasmid-encoded 16S rRNA yields an extra 19 or 39 nt, respectively. (B) Upper panel, polysome profile from cells growing in LB medium and expressing plasmid-derived ribosomes. Equivalent profiles were obtained for cells harboring either pASD^WT or pASD^mut. The 30S, 50S, 70S, and polysome peaks are indicated. Lower panel, primer extension analysis on equal amounts of RNAs from the fractions indicated on the profile. Quantification is presented in Table 1. ni, total RNA from cells in which the expression of plasmid-derived ribosomes was not induced. (C) Left panels, 2D electrophoresis of proteins from cells grown in LB medium and expressing rRNA from plasmid pASD^WT (upper panel) or pASD^mut (lower panel). Only parts of the gels are shown. Right panel, compared intensities of 178 Coomassie blue-stained spots from the two gels shown (abscissa, pASD^WT; ordinate, pASD^mut). The solid and dashed lines parallel to diagonal correspond to twofold and fourfold differences between the two gels, respectively. (D) Like ASD^WT ribosomes, ASD^mut ribosomes can translate bulk cellular mRNAs in the absence of cellular ribosomes, although the resulting protein pattern is not identical in both cases. Cells growing in minimal medium in the presence of arabinose were treated with spectinomycin (Spec.) and then pulse-chased with [³⁵S]methionine either immediately (0 min) or after 30 min. Asterisks and diamonds indicate proteins that are preferentially synthesized by ASD^WT or ASD^mut ribosomes, respectively. “Control” refers to cells carrying a pASD^WT derivative lacking the C1192T mutation conferring spectinomycin resistance. Molecular masses are given in kDa.

FIG. 4.

Histograms showing β-galactosidase activities from the indicated TIR-lacZ translational fusions in the presence of ASD^WT or ASD^mut ribosomes. β-Galactosidase units correspond to nanomoles of o-nitrophenyl-β-d-galactopyranoside hydrolyzed per min and per mg of total protein. The values shown are averages from at least three independent assays. (A) β-Galactosidase expression from the galE^WT and galE^CCUCC TIRs. (B) β-Galactosidase expression from various mutants of the rpsA TIR. The different TIRs contain either the wild-type GGA motifs (loopI+II^WT) (upper panel) or the mutant CCU motifs (loopI+II^mut) (lower panel) in loops I and II, together with the indicated additional mutations (see Fig. 1). Note the different scales of individual histograms. Error bars indicate standard deviations.

FIG. 5.

Compared patterns of lead(II)-induced cleavages in rpsA TIRs carrying either loopI+II^WT or loopI+II^mut (Fig. 1). (A) In vitro-transcribed wild-type (WT) and (loopI+II^mut,AUG) TIRs were probed with 60, 80, and 100 mM lead(II). Ctr, control without lead(II). L, alkaline hydrolysis ladder. Positions of the stem-and-loop regions of hairpins I and II and of single stranded regions (ss1 and ss2) are indicated. The smaller panels (right) focus on the apical loops of hairpins I and II; the relevant sequence is indicated on the right side, and uppercase is used for nucleotides from the loops. (B) The cleavage pattern of the region encompassing hairpins I and II is represented on the secondary structure model of the rpsA TIR (3). Strong, average, and weak cleavages are indicated by black, gray, and dotted arrows, respectively.