The nucleotide sequence of a leucine transfer RNA from E. coli

Abstract

We wish to report the nucleotide sequence of leucine transfer RNA (tRNALeu) from E. coli B.The particular tRNALeu labelled with 32P was first purified by electrophoresis in a polyacrylamide gel of RNA extracted from E. coli B grown in the presence of 32P-phosphate ( fig. 1). Examination of the various bands in the gel revealed that the particular leucine tRNA species was present in the slowest moving transfer RNA band (a), which in addition contains only one other, as yet, unknown tRNA. Final purification of the tRNALeu was achieved by chromatography on a benzoylated DEAE cellulose column [ 1 ] , the tRNALm eluting at a NaCl concentration around 0.7 M. The unknown tRNA species is eluted with 1 M NaCl containing 10% ethanol. Enzymatic digestions of the isolated tRNALeu and subsequent sequence determinations of the resulting oligonucleotides were carried out as previously described in detail by us [2][3][4] . Fig. 2 shows a two-dimensional fractionation of a ribonuclease T, digest of tRNALeu and an accompanying diagram giving the sequences of the nucleotides. A pancreatic ribonuclease digest was fractionated in a similar way and the sequences of the oligonucleotides determined. To overlap the end products large fragments were prepared by partial Tr or pancreatic ribonuclease digestions and separated by homochromatography [5]. The sequences of the partial fragments were determined from their complete T, and pancreatic ribonuclease products. From the large number of partial digestion products a unique sequence could be deduced ( fig. 3). As is the case for all other tRNAs known, the sequence can be arranged in the "clover leaf" pattern.The tRNA,,, consists of 87 nucleotides and is thus the largest tRNA sequenced so far. We have not 168 Fig. 1. Purification of tRNALeu by electrophoretic fractionation on a polyacrylamide gel. The gel (9.5% acrylamine, 0.5% bisacrylamide) was prepared as described by Adams et al.[lo] except that the buffer used was 0.09 M tris, boricsyid pH 8.3 containing 0.025 M EDTA. RNA labelled with P was isolated from E. co/i B grown in the presence of 32P-phosphate by phenol extraction. Electrophoresis was carried out at 40' for 16 hr at 350 V and 30 mA. North-Holland Publishing Company -Amsterdam

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 2. Species containing a large variable loop

“…The samples were adjusted to an absorbance of 100 at 260 nm so that the observed absorbance in the longer wavelength UV region directly reflects the percentage of the 260-nm extinction. The presence of 4-thiouridine was not detected during the sequencing of E. coli tRNALeU] (Dube et al, 1970), and we observe no 340-nm peak for this tRNA. Although Blank and Sol1 (1971) did not detect 4-thiouridine in tRNALeU2, they point out the need to confirm this with unlabeled tRNA due to the difficulty of identifying this residue with only very small amounts of labeled tRNA.…”

“…The final pure material accepted 1600 pmol of leucine per A260 unit; RNase T1 fingerprinting revealed its sequence to be that of E. coli tRNALe"! (Dube et al, 1970;Allaudeen et al, 1972).…”

Over Expression of a tRNALeu Isoacceptor Changes Charging Pattern of Leucine tRNAs and Reveals New Codon Reading

“…thiouridine (s 4 U), 5-methyluridine (m 5 U), 2 0 -O-methylguanosine (Gm), and an uncharacterized guanosine derivative 38,39. We monitored the eluate at 280 nm, at…”

Sequential processing of precursor tRNA molecules in Escherichia coli.

“…In the case of the multimeric precursor for tRNALeu (spot 4 in Fig. 2), ribothymidine, pseudouridine, and dihydrouridine are present but a methylated guanosine (17) at the 38th residue (from the 5' end) of tRNAPU is missing.…”