Pseudouridine-deficient transfer RNAs from Escherichia coli B and their use as substrates for pseudouridine synthetase.
Transfer RNAs isolated from Escherichia coli B grown in the presence of 2-thiouracil are deficient in pseudouridine. Much of this deficiency is from the T psi C region, which has only about 50% of its normal pseudouridine content. The other modified nucleoside from this region, ribothymidine, is reduced by only about 10%. Studies showed that 2-thiouracil is incoproated into the RNA of E. coli during growth in the presence of the analog. This incorporation appears to result from the replacement of uracil, occur in a random manner, and involve all RNA species. The extent of incorporation varies from 1 to 3 mol %, depending upon the preparation and RNA species examined. Electrophoresis on polyacrylamide gels and chromatography on Sephadex G-75 and reverse phase (Systen 5) columns of normal and 2-thiouracil-containing tRNAs revealed no profile differences. No accumulation of any precursor tRNA in the thiopyrimidine-treated cells is found. A partial recovery of the pseudouridine content of 2-thiouracil-containing tRNAs can be achieved in vivo by removal of the 2-thiouracil from the culture media. These transfer RNAs have also been used as substrates to study the properties of a partially purified preparation of pseudouridine synthetase II invitro and should be useful as substrates in the further purification of this enzyme.
D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D011560:Pseudouridine / Q000378:metabolism*; D012329:RNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012293:Rifampin / Q000494:pharmacology; D013889:Thiouracil / Q000494:pharmacology; D014529:Uridine / Q000031:analogs & derivatives*
D012329:RNA, Bacterial; D011560:Pseudouridine; D013889:Thiouracil; D012343:RNA, Transfer; D012293:Rifampin; D014529:Uridine