[An Escherichia coli strain producing a leaderless mRNA from the chromosomal lac promoter].

A special Escherichia coli strain capable of producing a leaderless lacZ mRNA from the chromosomal lac promoter was constructed to study the mechanism of leaderless mRNA translation. The translation efficiency of this noncanonical mRNA is very low in comparison with the canonical cellular templates, but it increases by one order of magnitude in the presence of chromosomal mutations in the genes encoding the ribosomal S1 and S2 proteins. The new strain possesses obvious advantages over the commonly used plasmid constructs (first of all, due to the constant dosage of lacZ gene in the cell) and opens possibilities for investigation of the specific conditions for leaderless mRNA translation in vivo using molecular genetic approaches.

[Extensive complementarity of the Shine-Dalgarno region and 3'-terminal sequence of 16S ribosomal RNA is inefficient for translation in vivo]. / Izbytochnaia komplementarnost' oblasti Shaina-Dal'garno i 3'-kontsevogo uchastka 16S ribosomnoi RNK neéffektivna pri transliatsii in vivo.

Translation initiation in Escherichia coli involves as a rule complementary interactions between a Shine-Dalgarno (SD) sequence upstream of the initiation codon and a highly conserved 3'-end sequence of 16S rRNA (anti-SD). The translation efficiency is believed to be directly affected by the affinity of the ribosome to the mRNA initiation region. Earlier, high-affinity RNA ligands to E. coli ribosomes were selected by the SELEX approach, with the ligands containing an extended SD-sequence well represented. In this work, we examined the ability of artificial ribosome binding sites (RBSs) containing such an extended (10-nt) SD-sequence (superSD) to drive translation in vivo, as well as its ability to form the translation initiation complex in vitro. Toe print experiments showed the formation of a ternary initiation complex on mRNA comprising superSD. Moreover, they proved the formation of an extended SD-duplex in the binary 30S-mRNA complex. Nevertheless, the superSD appeared to be inefficient in translation in vivo. We believe that the initiation complex involving a superSD-element is too stable to be functional; it may impede the transition from initiation to elongation, thus disrupting the transcription-translation coupling and inhibiting the formation of polysomes.