Protein-Precursor tRNA Contact Leads to Sequence-Specific Recognition of 5′ Leaders by Bacterial Ribonuclease P

JOURNAL OF MOLECULAR BIOLOGY

卷:396

Article

Koutmou, Kristin S.¹  Zahler, Nathan H.¹  Kurz, Jeffrey C.¹  Campbell, Frank E.^2,3  Harris, Michael E.^2,3  Fierke, Carol A.^1,4 

[1] Univ Michigan, Dept Chem, Ann Arbor, MI 48109 USA

[2] Case Western Reserve Univ, Ctr RNA Mol Biol, Cleveland, OH 44106 USA

[3] Case Western Reserve Univ, Dept Biochem, Cleveland, OH 44106 USA

[4] Univ Michigan, Dept Biol Chem, Sch Med, Ann Arbor, MI 48109 USA

关键词: RNase P;    sequence specificity;    tRNA processing;    substrate recognition;   

DOI  :  10.1016/j.jmb.2009.11.039

来源: Elsevier

【 摘 要 】

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 pretRNA 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 pretRNA 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. (C) 2009 Elsevier Ltd. All rights reserved.

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