RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells.
RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.
Kelly
K O
KO
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030-3305.
Reuven
N B
NB
Li
Z
Z
Deutscher
M P
MP
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010957:Plasmids; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000378:metabolism*; D012260:Ribonucleases / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D012322:RNA Precursors; D012365:RNA, Transfer, Tyr; D012343:RNA, Transfer; C056502:ribonuclease PH; D005095:Exoribonucleases; D012260:Ribonucleases; D001616:beta-Galactosidase