The residue immediately upstream of the RNase P cleavage site is a positive determinant

Brännvall, Mathias; Pettersson, B. M. Fredrik; Kirsebom, Leif A.

doi:10.1016/s0300-9084(02)01462-1

Cited by 40 publications

(72 citation statements)

References 62 publications

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“…Comparative sequence analysis supports a role for the nucleotide base at N(-1) in recognition by RNase P Previously, it was reported that tRNA genes from E. coli and Bacillus subtilis display a preference for U at position N(-1), and that few tRNA genes from E. coli encode a G at N(-1) Crary et al 1998;Brannvall et al 2002). Because A248 is conserved in bacterial and archaeal RNase P RNAs (Brown 1999), it follows that if this residue interacts with N(-1) in vivo, then this sequence preference should also be conserved in pre-tRNAs from Bacteria and Archaea.…”

Section: Conserved Residues In J5/15 Contribute To Ribozyme Affinity mentioning

confidence: 76%

“…Several studies have established that mutations at N(-1) affect RNase P function, and that a U at N(-1) is optimal Crary et al 1998;Loria and Pan 1998;Loria et al 1998;Zuleeg et al 2001;Brannvall et al 2002). Also, Kirsebom and coworkers have shown that in the context of an RNase P RNA with weak RCCA motif recognition, mutation of N(-2) can lead to miscleavage in a manner consistent with the proposed A248-N(-1) interaction (Brannvall et al 1998).…”

Section: Discussionmentioning

confidence: 92%

“…In the context of weakened interactions with the RCCA motif, cleavage site selection is dependent on the identity of N(-1) (Brannvall et al 1998(Brannvall et al , 2002. In addition, in the context of a minimal substrate representing only the acceptor stem of a tRNA, changes in N(-1) identity influence the rate of cleavage .…”

Section: Introductionmentioning

confidence: 99%

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Recognition of the 5′ leader of pre-tRNA substrates by the active site of ribonuclease P

Zahler¹,

Christian²,

Harris³

2003

RNA

148

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The bacterial tRNA processing enzyme ribonuclease P (RNase P) is a ribonucleoprotein composed of a ∼400 nucleotide RNA and a smaller protein subunit. It has been established that RNase P RNA contacts the mature tRNA portion of pre-tRNA substrates, whereas RNase P protein interacts with the 5 leader sequence. However, specific interactions with substrate nucleotides flanking the cleavage site have not previously been defined. Here we provide evidence for an interaction between a conserved adenosine, A248 in the Escherichia coli ribozyme, and N(-1), the substrate nucleotide immediately 5 of the cleavage site. Specifically, mutations at A248 result in miscleavage of substrates containing a 2 deoxy modification at N(-1). Compensatory mutations at N(-1) restore correct cleavage in both the RNA-alone and holoenzyme reactions, and also rescue defects in binding thermodynamics caused by A248 mutation. Analysis of pre-tRNA leader sequences in Bacteria and Archaea reveals a conserved preference for U at N(-1), suggesting that an interaction between A248 and N(-1) is common among RNase P enzymes. These results provide the first direct evidence for RNase P RNA interactions with the substrate cleavage site, and show that RNA and protein cooperate in leader sequence recognition.

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Section: Conserved Residues In J5/15 Contribute To Ribozyme Affinity mentioning

confidence: 76%

Section: Discussionmentioning

confidence: 92%

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Recognition of the 5′ leader of pre-tRNA substrates by the active site of ribonuclease P

Zahler¹,

Christian²,

Harris³

2003

RNA

148

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“…Likewise, B. subtilis RNase P RNA cleaved a ptRNA with a single 2Ј-deoxy C as the 5Ј-flank at a ϳ30-fold slower rate than a variant thereof with four additional ribonucleotides at the 5Ј-end (35). Exchanging the nt upstream of the cleavage site from U to A within an 8 -10 nt long 5Ј-flank reduced k cat in the M1 RNA-catalyzed reaction by a factor of 12-to almost 200-fold, depending on the substrate context (13). Even if one assumes that only a minor proportion of these reported reductions in catalytic rate took effect in the previous study (7), such effects on reactivity may easily explain the quantitative differences a 2Ј-deoxy modification at nt Ϫ1 had on the cleavage rate constant in the study by Smith and Pace (3400-fold) relative to the present study (ϳ150-fold, Fig.…”

Section: ј-Deoxy and 2ј-fluorinementioning

confidence: 99%

“…Studies with the model RNase P RNA ribozymes from Escherichia coli and Bacillus subtilis have indicated that at least two metal ions (the actual number influenced by the nature and concentration of monovalent cations present) play a specific role in productive enzyme-substrate complex formation and cleavage chemistry (7)(8)(9)(10)(11)(12)(13)(14). A solvent hydroxide or activated water molecule is assumed to be positioned by a metal ion for nucleophilic attack in an S N 2 in-line displacement mechanism (7,15).…”

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confidence: 99%

Catalysis by RNase P RNA

Persson

Cuzic

Hartmann

2003

Journal of Biological Chemistry

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Metal ions are essential cofactors for precursor tRNA (ptRNA) processing by bacterial RNase P. The ribose 2-OH at nucleotide (nt) ؊1 of ptRNAs is known to contribute to positioning of catalytic Me 2؉ . To investigate the catalytic process, we used ptRNAs with single 2-deoxy (2-H), 2-amino (2-N), or 2-fluoro (2-F) modifications at the cleavage site (nt ؊1). 2 modifications had small (2.4 -7.7-fold) effects on ptRNA binding to E. coli RNase P RNA in the ground state, decreasing substrate affinity in the order 2-OH > 2-F > 2-N > 2-H. Effects on the rate of the chemical step (about 10-fold for 2-F, almost 150-fold for 2-H and 2-N) were much stronger, and, except for the 2-N modification, resembled strikingly those observed in the Tetrahymena ribozyme-catalyzed reaction at corresponding position. Mn 2؉ rescued cleavage of the 2-N but also the 2-H-modified ptRNA, arguing against a direct metal ion coordination at this location. Miscleavage between nt ؊1 and ؊2 was observed for the 2-N-ptRNA at low pH (further influenced by the base identities at nt ؊1 and ؉73), suggesting repulsion of a catalytic metal ion due to protonation of the amino group. Effects caused by the 2-N modification at nt ؊1 of the substrate allowed us to substantiate a mechanistic difference in phosphodiester hydrolysis catalyzed by Escherichia coli RNase P RNA and the Tetrahymena ribozyme: a metal ion binds next to the 2 substituent at nt ؊1 in the reaction catalyzed by RNase P RNA, but not at the corresponding location in the Tetrahymena ribozyme reaction.Endonucleolytic 5Ј maturation of tRNA primary transcripts is catalyzed by the ribonucleoprotein enzyme ribonuclease P (RNase P) 1 in all three domains of life (Archaea, Bacteria, and Eukarya) as well as in mitochondria and chloroplasts (1-3). Bacterial RNase P enzymes are composed of a catalytic RNA subunit (4), ϳ400 nucleotides (nt) in length, and a single small protein of typically 120 amino acids (5). The only known exception may be the hyperthermophilic bacterium Aquifex aeolicus.Neither genes for RNase P RNA (rnpB) or protein (rnpA) subunits could be identified in the sequenced genome, nor have biochemical studies provided evidence for the presence of a bacterial-like RNase P activity (6).Processing of ptRNAs by RNase P results in cleavage products with 3Ј-OH and 5Ј-phosphate termini. Studies with the model RNase P RNA ribozymes from Escherichia coli and Bacillus subtilis have indicated that at least two metal ions (the actual number influenced by the nature and concentration of monovalent cations present) play a specific role in productive enzyme-substrate complex formation and cleavage chemistry (7-14). A solvent hydroxide or activated water molecule is assumed to be positioned by a metal ion for nucleophilic attack in an S N 2 in-line displacement mechanism (7, 15). A 2Ј-deoxyribose substitution at the RNase P cleavage site was previously reported to affect binding of catalytically important Mg 2ϩ and to substantially reduce the rate of catalysis by E. coli RNase P RNA (7, 16 -18). Recent ...

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Importance of RNA‐protein interactions in bacterial ribonuclease P structure and catalysis

Smith¹,

Hsieh²,

Fierke³

2007

Biopolymers

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Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) complex that catalyzes the metal‐dependent maturation of the 5′ end of precursor tRNAs (pre‐tRNAs) in all organisms. RNase P is comprised of a catalytic RNA (P RNA), and at least one essential protein (P protein). Although P RNA is the catalytic subunit of the enzyme and is active in the absence of P protein under high salt concentrations in vitro, the protein is still required for enzyme activity in vivo. Therefore, the function of the P protein and how it interacts with both P RNA and pre‐tRNA have been the focus of much ongoing research. RNA‐protein interactions in RNase P serve a number of critical roles in the RNP including stabilizing the structure, and enhancing the affinity for substrates and metal ions. This review examines the role of RNA‐protein interactions in bacterial RNase P from both structural and mechanistic perspectives. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 329–338, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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The residue immediately upstream of the RNase P cleavage site is a positive determinant

Cited by 40 publications

References 62 publications

Recognition of the 5′ leader of pre-tRNA substrates by the active site of ribonuclease P

Recognition of the 5′ leader of pre-tRNA substrates by the active site of ribonuclease P

Catalysis by RNase P RNA

Importance of RNA‐protein interactions in bacterial ribonuclease P structure and catalysis

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