[Regulation of synthesis of ribosomal protein L7-L12: role of intercistronic rplJL region as an enhancer of translation]. / Reguliatsiia sinteza ribosomnogo belka L7/12: rol' mezhtsistronnoi oblasti rplJL kak usilitelia transliatsii.

The ability of the Escherichia coli intercistronic rplJL region to initiate effectively the synthesis of the ribosomal protein L7/12, the only ribosomal component present in the ribosome in four copies rather than in one was studied in vivo and in vitro. It was shown that the structural determinants located upstream from the Shine-Dalgarno sequence and sharing structural motifs with the known E. coli translational enhancers are necessary for high activity of this region in translation initiation. These data indicate that mRNA-protein interactions through the ribosomal S1 protein play an important role in the formation of the initiation complex, and an enhancer region within the leader of the L7/12 mRNA serves as a target for this protein.

RNA folding kinetics regulates translation of phage MS2 maturation gene.

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem-loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3' moiety contains the Shine-Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem-loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.

[Analysis of the secondary structure of the regulatory region of mRNA of the Escherichia coli rpsA gene]. / Analiz vtorichnoi struktury reguliatornoi oblasti mRNK gena rpsA Escherichia coli.

The rpsA gene of E. coli coding for the ribosomal protein S1 was inserted into the plasmid pGEM-3Z under the control of a T7 promoter. The resulted plasmid was used for mRNA preparation in vitro. The toeprint analysis of the rpsA mRNA revealed a strong S1 dependence: 30S ribosomal subunits lacking S1 were inactive in the 30S initiation complex formation; addition of the free S1 restored subunits' ability to bind mRNA; a molar excess of the free S1 over ribosomes was inhibitory. The secondary structure of the rpsA mRNA in the vicinity of the initiation codon was probed with the use of specific ribonucleases. Basing on the experimental data obtained we suggest a model for the structural organisation of the rpsA mRNA translation initiation region.