Sequential processing of precursor tRNA molecules in Escherichia coli.

Abstract

In a temperature-sensitive mutant of E. cofl defective in tRNA biosynthesis, many tRNA precursors, including monomeric and multimeric forms, accumulate. Some of the multimeric precursors contain three or more tRNA seuences within a molecule. These large precursors were cleaved by cell extracts first into intermediate size pieces which were subsequently processed by RNase P. On the basis of heat stability of mutant cell extracts, the endonuclease responsible for the initial cleavage appears to be distinct from RNase P and is designated RNase 0. One of the monomeric precursors was shown to be processed first by RNase P and the product subsequently cleaved further into a smaller molecule. The nuclease responsible for this second cleavage also appears to be distinct rom RNase P and is designated RNase Q. The functions of these nucleases are sequential in the trimming process with respect to that of RNase P; RNase 0 works prior to RNase P and RNase Q after RNase P but in both cases, not vice versa. The notion that tRNAs are formed from the initial transcripts of tRNA genes via a series of processing steps has been well accepted (1). These initial precursors are expected to be larger than mature tRNA size. Thus, the precursor molecules must be cleaved at specific sites in the maturation process. In the case of Su3+ suppressor tRNA, an unmodified precursor RNA carrying additional nucleotides at both the 5' and 3' termini has been detected (2, 3), and an endonuclease that cleaves this precursor at the 5' end of Su3+ tRNA has been identified in Escherichia coli extract and designated RNase P (4). Temperature-sensitive mutants of E. coli defective in this nucleolytic activity have been reported (5-7). So far, this is the only nuclease that has been clearly shown to participate in the trimming process of E. colh. It is anticipated, however, that some other nucleases are also required in maturation of tRNA molecules. Although a few other candidates have been suggested (1), none of them has been as firmly established as RNase P. Thus it remains to be solved how many nucleolytic enzymes participate in the processing of E. coli tRNAs and how they work during the trimming process. In the present paper we report that there are at least two other nucleases in E. coil that are required for tRNA biosynthesis. Evidence will be presented showing that these two nucleases and RNase P function in a highly ordered fashion on the precursors of E. coli tRNA molecules. MATERIALS AND METHODSBacterial Strains. E. coli 4273 (su, lacam, T6amr, BF23amr) and TS241, a temperature-sensitive mutant of 4273, defective in tRNA biosynthesis were described previously (6). E. colh Q13 (RNase I-) was originally from Dr. R. F. Gesteland.Preparation of RNA and Polyacrylamide Gel Electrophoresis. Low phosphate medium used for 32p labeling and extraction of RNA were described earlier (6). Electrophoresis in 10% or 12% polyacrylamide gel was performed in 0.1 M Tris-acetate (pH 8.3) as described by DeWachter and Fiers (9). 20% polyacrylamide gel was pre...

RNA Processing and the Intervening Sequence Problem

“…The RNase pts mutants have allowed the characterization of a large number of tRNA precursors. When E. coli carrying an RNase pts mutation is labeled with 32P04 at the nonpermissive temperature more than 30 tRNA precursors accumulate (31)(32)(33)(34). These can be separated by polyacrylamide gel electrophoresis and further characterized.…”

Processing of procaryotic ribonucleic acid.

“…Because in all E. coli tRNA's examined, the CCA terminus of the mature molecule is encoded in the DNA and is present in the RNA precursor, only the activity of RNase D would be required for 3' trimming. It has been suggested, however, that RNase D trimming in vivo or in vitro may require prior RNase P cleavage at the 5' side of the precursor tRNAIT (61,142). In a temperature-sensitive RNase P strain, this and many other pre-tRNA's are found to bear extra sequences past their mature 3' termini (6,159).…”

Characterization of a Salmonella typhimurium hisU mutant defective in tRNA precursor processing

“…For instance, phage T4 during infection ofEscherichia coli cells induces the biosynthesis of phage-coded tRNAs whose dimeric precursors have a relatively long half-life, so their structure as well as some of their properties could be determined (11,12,17,18,26). A considerable amount of information has also been obtained by studying some tRNATy,su3+ precursors which show a slow maturation as a consequence of mutations in their nucleotide sequence (2,29) or mutations in maturation-specific nucleases (22,24). The isolation of these two types of mutants is based on the same principle: selection of conditional lethal mutants which, at a nonpermissive temperature, will not synthesize mature tRNA molecules able to function in protein synthesis.…”