Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Abstract

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met). Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.

Fig. 1. Rate of relative toeprints obtained with leaderless cI mRNA (A), cISD mRNA (B) and ompA mRNA (C) in the presence (filled symbols) and absence (open symbols) of IF2. The kinetic toeprinting reactions contained 2 pmol of 30S subunits, 5 pmol of IF2 (when present), 0.8 pmol of fMet-tRNA_f^Met and 1 mM GTP, and were pre-incubated for 20 s before the mRNA(s) was added for the times (given in seconds) indicated. The toeprint reaction was then started with MMLV as described in Materials and methods.

Fig. 2. Selective stimulation of CI synthesis by IF2. (A–D) In vitro translation of equimolar amounts of cI and ompA mRNA, respectively, with an E.coli S100 extract as described in Materials and methods. The different ratios of IF2 to added 70S ribosomes are indicated above the panels. The ratio of IF1 or IF3 to 70S was 0.2:1 in all reactions. The samples were loaded on a 12% SDS–polyacrylamide gel and the rate of CI and OmpA synthesis at different molar ratios of IF2 to 70S ribosomes was calculated by ImageQuant analysis (B and D). In the absence of exogenously added IF2 (A and C, lane 1), the CI and OmpA synthesis rate was set to 1. (E and F) In vitro translation competition assay with cI and ompA mRNA at different molar IF2:70S ribosome ratios. The conditions were as in (A–D), except that the in vitro translation system was programmed with equimolar amounts of the two mRNAs. The CI and OmpA synthesis rate is set to 1 at a 0.2:1 molar ratio of IF1, IF2 or IF3 to ribosomes (E, lane 2).

Fig. 3. Selection of the 5′-terminal and the internal AUG on ompAΔ117 mRNA in the presence of IF2. (A) Depiction of the ompAΔ117 mRNA. The start codon(s) and the authentic SD sequence of ompA mRNA are indicated by bars. For ompAΔ117, two toeprint signals are obtained, which result from formation of a ternary complex over AUG1 (nucleotide –20 to –18) and AUGi (nucleotide 1–3), which represents the authentic start codon of ompA mRNA. The positions of the toeprint signals are indicated by filled (AUG1) and open circles (AUGi). (B) Kinetic toeprints on ompAΔ117 mRNA in the presence and absence of IF2. The final concentration of 30S subunits, IF2, fMet-tRNA_f^Met and ompAΔ117 mRNA was 0.5, 1, 0.2 and 0.005 pmol/µl, respectively. Lane 1, primer extension in the absence of 30S subunits and fMet-tRNA_f^Met. Lanes 2–6, kinetic toeprint analysis in the absence of IF2. Lanes 7–11, kinetic toeprint analysis in the presence of IF2. 30S subunits, fMet-tRNA_f^Met, IF2 and mRNA were incubated for different times (given in seconds) as indicated at the top. Lane 12, G sequencing reaction. The toeprinting signals obtained for AUG1 and AUGi are indicated by a filled and open circle, respectively. (C) Relative toeprints on ompAΔ117 mRNA for AUG1 and AUGi. The relative toeprints at either start codon obtained in the absence of IF2 were normalized to that of AUG1 and AUGi obtained in the presence of IF2 after 80 s of incubation.

Fig. 4. In vivo expression of cI–lacZ and ompA in the presence of elevated concentrations of IF2. (A) Increased intracellular concentrations of IF2 stimulate cI–lacZ expression. β-galactosidase measurements were performed at various times after heat induction of the infB gene. White and black bars represent the β-galactosidase synthesis obtained with strain UB89-1 (pKTplaccI/pinfB) at 28 and 43°C, respectively. The β-galactosidase activity obtained with strain UB89-1 (pKTplaccI/plc2833) at 28 and 43°C is indicated by gray and hatched bars, respectively. The cultures were grown at 28°C and shifted to 43°C at an OD₆₀₀ of 0.3. The β-galactosidase values are given in Miller units. (B) Determination of OmpA synthesis at basal and increased concentrations of IF2. Aliquots of strain UB89-1 (pKTplaccI/pinfB) were withdrawn at 28 or 43°C at the indicated OD values; 0.1 OD₆₀₀ units of total cellular protein were loaded on to a 12% SDS–polyacrylamide gel for a given OD₆₀₀ value. OmpA synthesis was determined by quantitative immunoblotting as described in Materials and methods. Black and white bars correspond to OmpA synthesis at 43 and 28°C, respectively. (C) Plasmid pinfB-directed synthesis of IF2. –, no induction of the plasmid-borne infB gene; +, heat induction of the infB gene at an OD₆₀₀ of 0.3. As described in (B), aliquots of strain UB89-1 (pKTplaccI/pinfB) were withdrawn at OD₆₀₀ values of 0.3, 0.4, 0.6 and 0.8. For a given OD, 0.1 OD₆₀₀ unit of total cellular protein was subjected to SDS–PAGE and subsequent quantitative immunoblotting as described in Material and methods. Only the relevant section of the immunoblot showing the IF2-specific band(s) is depicted.

Fig. 5. Translation of equimolar amounts of cI and ompA mRNA in the E.coli S30, rabbit reticulocyte and S.solfataricus in vitro translation systems. Samples from the in vitro translation reactions were analyzed on a 12% SDS–polyacrylamide gel. Translation of ompA mRNA with the E.coli S30 extract, reticulocyte system and S.solfataricus system is shown in lanes 1, 4 and 7, respectively. Translation of cI mRNA with the E.coli S30 extract, reticulocyte system and S.solfataricus system is shown in lanes 2, 5 and 8, respectively. Translation of cI_AUG1→CUG mRNA with the E.coli S30 extract, reticulocyte system and S.solfataricus system is shown in lanes 3, 6 and 9, respectively. The positions of the bands corresponding to CI and OmpA are marked. The band marked with a filled circle in lane 5 results from an internal translation event in cI mRNA (see text).

Fig. 6. Translational initiation pathways in prokaryotes and eukaryotes. (A) Recruitment of the prokaryotic ribosome by an mRNA containing a canonical rbs through the SD–anti-SD interaction in the absence of P-site-bound initiator tRNA. (B) Recognition of leaderless mRNA by a prokaryotic 30S ribosome–initiator tRNA complex. IF2 is depicted by a closed circle. (C) Recognition of the cap complex by an eukaryotic 40S ribosome–initiator tRNA complex (reviewed by Gallie, 1998). The cap-binding complex eIF4F, consisting of eIF4E (crescent), eIF4G (oval), eIF4A (circle) and eIF4B (small oval); eIF3 (black square) and eIF2 (closed circle) are also depicted.