In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fmet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entry channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

The study of translation initiation in prokaryotes assumes that there should be a mechanism different from the canonical model, which postulates the formation of the pre-initiation complex through the interaction of the Shine-Dalgarno sequence (SD) at the 5\'-end of mRNA and the anti-Shine-Dalgarno site at the 3\'-end of 16S rRNA. In this paper we\'ve studied the effect of TPS (Translation-initiation Promoting Site) on β-glucuronidase expression in E. coli cells at different cultivation temperatures. The examined leader sequences were cloned into the pET23c plasmid upstream the β-glucuronidase gene; protein expression was performed in E. coli BL21 (DE3) cells. β-glucuronidase activity was measured in bacterial cell extracts via paranitrophenyl b-D-glucuronide assay. The quantity of expressed protein was measured by Western blotting with following densitometry. It was shown that TPS increases the level of protein expression at stressful conditions (10 °C and 44 °C) 5-8 times compared to control. The combination of TPS and SD sites in the 5\'-leader sequence of the mRNA created an enhancer that increased the expression level 2-3.6 times compared to a single SD-sequence. Based on the obtained data and the computer modeling of interaction between 16S rRNA and TPS, we proposed an alternative variation of prokaryotic translation initiation.

Translation of mRNAs is the primary function of the ribosomal machinery. Although cells allow for a certain level of translational errors/mistranslation (which may well be a strategic need), maintenance of the fidelity of translation is vital for the cellular function and fitness. The P-site bound initiator tRNA selects the start codon in an mRNA and specifies the reading frame. A direct P-site binding of the initiator tRNA is a function of its special structural features, ribosomal elements, and the initiation factors. A highly conserved feature of the 3 consecutive G:C base pairs (3 GC pairs) in the anticodon stem of the initiator tRNAs is vital in directing it to the P-site. Mutations in the 3 GC pairs diminish/abolish initiation under normal physiological conditions. Using molecular genetics approaches, we have identified conditions that allow initiation with the mutant tRNAs in Escherichia coli. During our studies, we have uncovered a novel phenomenon of in vivo initiation by elongator tRNAs. Here, we recapitulate how the cellular abundance of the initiator tRNA, and nucleoside modifications in rRNA are connected with the tRNA selection in the P-site. We then discuss our recent finding of how a conserved feature in the mRNA, the Shine-Dalgarno sequence, influences tRNA selection in the P-site.