[Correlation-regression analysis of the impact of motor transport on human health].

Statistical analysis of the time series and spatial data of sociohygienic monitoring has yielded models of simple and multiple linear regressions, which reflect the impact of motor transport on human health, their statistical stability. The morbidity rates have ranked by the response to the changing values of motor transport. A contribution of environmental factors to the morbidity rates has been determined. Areas of application of built models are proposed.

Short-range RNA-RNA crosslinking methods to determine rRNA structure and interactions.

We describe details of procedures to analyze RNA-RNA crosslinks made by far-UV irradiation (< 300 nm) or made by irradiation with near-UV light (320-365 nm) on RNA containing photosensitive nucleotides, in the present case containing 4-thiouridine. Zero-length crosslinks of these types must occur because of the close proximity of the participants through either specific interactions or transient contacts in the folded RNA structure, so they are valuable monitors of the conformation of the RNA. Procedures to produce crosslinks in the 16S ribosomal RNA and between the 16S rRNA and mRNA or tRNA are described. Gel electrophoresis conditions are described that separate the products according to their structure to allow the determination of the number and frequency of the crosslinking products. Gel electrophoresis together with an ultracentrifugation procedure for the efficient recovery of RNA from the polyacrylamide gels allows the purification of molecules containing different crosslinks. These separation techniques allow the analysis of the sites of crosslinking by primer extension and RNA sequencing techniques. The procedures are applicable to other types of RNA molecules with some differences to control levels of crosslinking and separation conditions.

UV-induced crosslinks in the 16S rRNAs of Escherichia coli, Bacillus subtilis and Thermus aquaticus and their implications for ribosome structure and photochemistry.

Sixteen long-range crosslinks are induced in Escherichia coli 16S rRNA by far-UV irradiation. Crosslinking patterns in two other organisms, Bacillus subtilis and Thermus aquaticus, were investigated to determine if the number and location of crosslinks in E.coli occur because of unusually photoreactive nucleotides at particular locations in the rRNA sequence. Thirteen long-range crosslinks in B.subtilis and 15 long-range crosslinks in T.aquaticus were detected by gel electrophoresis and 10 crosslinks in each organism were identified completely by reverse transcription analysis. Of the 10 identified crosslinks in B.subtilis, eight correspond exactly to E.coli crosslinks and two crosslinks are formed close to sites of crosslinks in E.coli. Of the 10 identified crosslinks in T.aquaticus, five correspond exactly to E.coli crosslinks, three are formed close to E.coli crosslinking sites, one crosslink corresponds to a UV laser irradiation-induced crosslink in E.coli and the last is not seen in E.coli. The overall similarity of crosslink positions in the three organisms suggests that the crosslinks arise from tertiary interactions that are highly conserved but with differences in detail in some regions.

Initiation factor 3-induced structural changes in the 30 S ribosomal subunit and in complexes containing tRNA(f)(Met) and mRNA.

Initiation factor 3 (IF3) acts to switch the decoding preference of the small ribosomal subunit from elongator to initiator tRNA. The effects of IF3 on the 30 S ribosomal subunit and on the 30 S.mRNA. tRNA(f)(Met) complex were determined by UV-induced RNA crosslinking. Three intramolecular crosslinks in the 16 S rRNA (of the 14 that were monitored by gel electrophoresis) are affected by IF3. These are the crosslinks between C1402 and C1501 within the decoding region, between C967xC1400 joining the end loop of a helix of 16 S rRNA domain III and the decoding region, and between U793 and G1517 joining the 790 end loop of 16 S rRNA domain II and the end loop of the terminal helix. These changes occur even in the 30 S.IF3 complex, indicating they are not mediated through tRNA(f)(Met) or mRNA. UV-induced crosslinks occur between 16 S rRNA position C1400 and tRNA(f)(Met) position U34, in tRNA(f)(Met) the nucleotide adjacent to the 5' anticodon nucleotide, and between 16 S rRNA position C1397 and the mRNA at positions +9 and +10 (where A of the initiator AUG codon is +1). The presence of IF3 reduces both of these crosslinks by twofold and fourfold, respectively. The binding site for IF3 involves the 790 region, some other parts of the 16 S rRNA domain II and the terminal stem/loop region. These are located in the front bottom part of the platform structure in the 30 S subunit, a short distance from the decoding region. The changes that occur in the decoding region, even in the absence of mRNA and tRNA, may be induced by IF3 from a short distance or could be caused by the second IF3 structural domain.

Distribution of cross-links between mRNA analogues and 16 S rRNA in Escherichia coli 70 S ribosomes made under equilibrium conditions and their response to tRNA binding.

The interaction between mRNA and Escherichia coli ribosomes has been studied by photochemical cross-linking using mRNA analogues that contain 4-thiouridine (s4U) or s4U modified with azidophenylacyl bromide (APAB), either two nucleotides upstream or eight nucleotides downstream from the nucleotide sequence ACC, the codon for tRNA(Thr). The sequences of the mRNA analogues were described earlier (Stade, K., Rinke-Appel, J., and Brimacombe, R. (1989) Nucleic Acids Res. 17, 9889-9908; Rinke-Appel, J., Stade, K., and Brimacombe, R. (1991) EMBO J. 10, 2195-2202). Under equilibrium conditions, both of these mRNA analogues bind and cross-link to 70 S ribosomes without the presence of tRNA(Thr); however, there are significant increases both in binding and particularly in cross-linking in the presence of the tRNA(Thr). Four regions contain cross-linking sites that increase in the presence of tRNA, C1395, A532, A1196 (and minor sites around these three positions), and C1533/U1532. Three other cross-linking sites, U723, A845, and U1381, show very little change in extent of cross-linking when tRNA is present. A conformational change in the 30 S subunit allowing additional accessibility to the 16 S rRNA by the mRNA analogues upon tRNA binding best explains the behavior of the tRNA-dependent and tRNA-independent mRNA-16 S rRNA cross-linking sites.

Three dimensional model for the 16S ribosomal RNA that incorporates information for the mRNA track.

A three dimensional model for the 16S rRNA in the ribosome is described that accommodates information for mRNA.16S rRNA interactions as well as accommodating information that has been used as constraints for determining the internal 16S rRNA structure. mRNA.16S rRNA interactions have been summarized from experiments that employ photoaffinity crosslinking to identify sites at which the mRNA comes into close contact with the 16S rRNA. The mRNA track that has been constructed follows a path completely around the middle of the 30S subunit, occupying a space above 16S rRNA domains I and II and below 16S rRNA domain III. The mRNA track contains regions associated with contacts with the 16S rRNA in, and just upstream of, the P tRNA site, associated with contacts around the A site and associated with neighborhoods upstream of the Shine-Dalgarno region and downstream of the decoding region. Structural constraints that come from UV-induced RNA-RNA crosslinks, suggest that the mRNA decoding region lies in a groove between three 16S rRNA duplex regions facing the 50S subunit.

Puromycin reaction of the A-site bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.

Puromycin reaction for the A site-bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) synthesized in 70S ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. A detailed study of the kinetics of the puromycin reaction, its comparison with that of spontaneous translocation, the use of antibiotic viomycin as an effective inhibitor of spontaneous translocation revealed that, besides spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction per se triggers conformational changes in the ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl-site of the peptidyltransferase center. This is detected functionally as the ability of such an A site bound peptidyl-tRNA to react with puromycin. This reaction is highly pronounced at elevated (25 degrees C) temperature but can be hardly detected at 0 degrees C.