Nuclear pre-tRNA terminal structure and RNase P recognition.
Nuclear pre-tRNA transcripts often contain an extension of the aminoacyl stem formed by base pairing between the 5'-leader and 3'-trailing sequences, but the -1 position preceding the mature 5' end is usually left unpaired. Considering recently proposed tertiary structural models for RNase P RNAs, we hypothesize that the -1 mismatch prevents a strong, coaxially extended aminoacyl stem, which might otherwise sterically interfere with substrate positioning in the RNase P active site. This hypothesis is tested by creating uninterrupted aminoacyl stem extensions in four nuclear tRNA precursors that normally have a mismatched nucleotide at position -1, and comparing their cleavage rates with those of the normal precursors. Determinations of Km and kcat values for a normal and an altered pre-tRNA(SUP53), which exhibits the most subtle structural alteration immediately upstream of the cleavage site, indicate that the mismatch at position -1 is an important structural requirement for both substrate affinity and efficient catalysis (and/or product release) by nuclear RNase P. This conclusion is further supported in vivo, where the pre-tRNA(SUP53) mutant precursor lacking the -1 mismatch is shown to accumulate.
Lee
Y
Y
Department of Biological Chemistry, The University of Michigan, Ann Arbor 48109-0606, USA.
Kindelberger
D W
DW
Lee
J Y
JY
McClennen
S
S
Chamberlain
J
J
Engelke
D R
DR
D004722:Endoribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry* / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D043262:Ribonuclease P; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D012322:RNA Precursors; D016337:RNA, Catalytic; D012331:RNA, Fungal; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P