RNA PROTEIN CROSSLINKS INTRODUCED INTO E. coli RIBOSOMES BY USE OF THE INTRINSIC PROBE 4-THIOURIDINE

Abstract— 70S Ribosome substituted by the uridine photoactivable analogue 4-thiouridine has been prepared by an in vivo method (substitution level 4.5%). The r-proteins crosslinked to 16S and 23S rRNA before and after 366-nm photoactivation were identified. Proteins S2-S7-S9/11-S18 are found linked to 16S RNA in dark-prepared 30S subunits. Illumination increases uniformly their binding by a factor of 2.5. Similarly, proteins L5-L15-L18-L23-L28-L32 are found crosslinked to 23S RNA in dark-prepared 50S subunits. Photoactivation increases their binding but in addition promotes the covalent linking of proteins L1-L3-L4.     

METABOLISM OF tRNAs IN GROWING CELLS OF Escherichia coli ILLUMINATED WITH NEAR-ULTRAVIOLET LIGHT

Abstract— The tRNA metabolism which accompanies illumination of growing E. coli cells has been examined in conditions that led to growth delay. (i) The in vivo formation of the 8–13 link was followed by a fluorimetric procedure and revealed pseudo-first order kinetics very close to those obtained in vim under the same illumination conditions. The yield of 8–13 link appears to be quantitative (± 10%). Comparison of these kinetics with the radiochromatographic data of Blanchetot et al . (1984) suggests the transient formation during illumination of a new RNase-T,-resistant dinucleotide in tRNA distinct from the 8–13 link. (ii) Evidence is provided that under illumination some tRNA molecules lack one or more bases in a specific position in the sequence, thus yielding discrete fragments after aniline treatment. (iii) During the growth lag, uracil incorporation into nucleic acids occurs at an apparent rate between 4–8% of that normally observed during exponential growth. Evidence is provided however that the pyrimidine ribonucleoside triphosphate pools are strongly perturbed after illumination. Comparison of exogenous [³H]uracil incorporation into two strains proficient or deficient in uracil biosynthesis suggests a derepression of the endogenous path after light treatment. In addition, the UTP-to-CTP conversion is inhibited. In spite of preferential incorporation of exogenously labelled uracil in tRNA after illumination, a possible pyrimidine base turnover cannot be proved. These data are compatible with tRNA repair (Blanchetot et al ., 1984) involving a few tRNA species.