Protein-precursor tRNA contact leads to sequence-specific recognition of 5' leaders by bacterial ribonuclease P.
Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5' leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5' side of the cleavage site (N(-4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(-4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(-4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(-4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(-4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(-4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(-4) that enhances RNase P affinity. This observation suggests that specificity at N(-4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.
Koutmou
Kristin S
KS
Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, MI 48109, USA.
Zahler
Nathan H
NH
Kurz
Jeffrey C
JC
Campbell
Frank E
FE
Harris
Michael E
ME
Fierke
Carol A
CA
D020121:5' Untranslated Regions / Q000235:genetics*; D000241:Adenosine / Q000378:metabolism; D019943:Amino Acid Substitution / Q000187:drug effects; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D012322:RNA Precursors / Q000378:metabolism*; D012343:RNA, Transfer / Q000235:genetics; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects
D020121:5' Untranslated Regions; D050505:Mutant Proteins; D009711:Nucleotides; D012322:RNA Precursors; D012343:RNA, Transfer; D043262:Ribonuclease P; D000241:Adenosine; D002118:Calcium