Function of heterologous and truncated RNase P proteins in <i>Bacillus subtilis</i>

Gößringer, Markus; Hartmann, Roland K.

doi:10.1111/j.1365-2958.2007.05962.x

Cited by 17 publications

(10 citation statements)

References 48 publications

(83 reference statements)

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“…When divergences are further corrected to account for unseen substitutions, the sequences we tested exhibit pairwise divergences that range from 0.27 ( Proteus mirabilis rnpA ) to 1.72 ( S. oralis rnpA) amino acid substitutions per site. We thus confirm and extend the finding that endogenous bacterial RNase P proteins can be complemented in vivo by diverse phylogenetic heterologs [20], supporting tertiary structure and function conservation as defining features of the evolution of bacterial rnpA proteins.…”

Section: Resultssupporting

confidence: 83%

“…Comparative studies suggest that the tertiary structures of bacterial RNase P protein show remarkable conservation despite significant divergence in primary sequence [15], [16], [17], [18], [19]. As a result, highly divergent protein subunits taken from phylogenetically distant sources can still form a functional holoenzyme in vivo with the RNase P RNA subunit of Bacillus subtilis [17], [20].…”

Section: Introductionmentioning

confidence: 99%

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By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

Turrini¹,

Loveland²,

Dorit

2012

PLoS ONE

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Bacterial RNase P is an essential ribonucleoprotein composed of a catalytic RNA component (encoded by the rnpB gene) and an associated protein moiety (encoded by rnpA ). We construct a system that allows for the deletion of the essential endogenous rnpA copy and for its simultaneous replacement by a heterologous version of the gene. Using growth rate as a proxy, we explore the effects on fitness of heterologous replacement by increasingly divergent versions of the RNase P protein. All of the heterologs tested complement the loss of the endogenous rnpA gene, suggesting that all existing bacterial versions of the rnpA sequence retain the elements required for functional interaction with the RNase P RNA. All replacements, however, exact a cost on organismal fitness, and particularly on the rate of growth acceleration, defined as the time required to reach maximal growth rate. Our data suggest that the similarity of the heterolog to the endogenous version — whether defined at the sequence, structure or codon usage level — does not predict the fitness costs of the replacement. The common assumption that sequence similarity predicts functional similarity requires experimental confirmation and may prove to be an oversimplification.

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Section: Resultssupporting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

Turrini¹,

Loveland²,

Dorit

2012

PLoS ONE

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“…Therefore, we addressed the question whether Hera and RnpA contact the same or overlapping binding sites on RNase P RNA in competition experiments (Figure 5). Cross-species complementation between different RNase P RNAs and RnpA proteins has been demonstrated in general (16,23,39,40), and specifically for T. thermophilus RNase P RNA and E. coli RnpA used here (41). Donor/acceptor-labeled Hera (50 pM) was incubated with 3 mM ADPNP and 50 nM T. thermophilus RNase P RNA, a concentration sufficient to induce the closure of the inter-domain cleft, and 200 nM E. coli RnpA (4-fold excess over Rnase P RNA) was added.…”

Section: Resultsmentioning

confidence: 80%

The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

Linden¹,

Hartmann²,

Klostermeier³

2008

Nucleic Acids Research

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DEAD box helicases use the energy of ATP hydrolysis to remodel RNA structures or RNA/protein complexes. They share a common helicase core with conserved signature motifs, and additional domains may confer substrate specificity. Identification of a specific substrate is crucial towards understanding the physiological role of a helicase. RNA binding and ATPase stimulation are necessary, but not sufficient criteria for a bona fide helicase substrate. Here, we report single molecule FRET experiments that identify fragments of the 23S rRNA comprising hairpin 92 and RNase P RNA as substrates for the Thermus thermophilus DEAD box helicase Hera. Both substrates induce a switch to the closed conformation of the helicase core and stimulate the intrinsic ATPase activity of Hera. Binding of these RNAs is mediated by the Hera C-terminal domain, but does not require a previously proposed putative RNase P motif within this domain. ATP-dependent unwinding of a short helix adjacent to hairpin 92 in the ribosomal RNA suggests a specific role for Hera in ribosome assembly, analogously to the Escherichia coli and Bacillus subtilis helicases DbpA and YxiN. In addition, the specificity of Hera for RNase P RNA may be required for RNase P RNA folding or RNase P assembly.

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“…In fact, a genetic complementation approach revealed that both type A and M archaeal RPPs (expressed either individually or as binary complexes) were inadequate in rescuing the bacterial RPP defect in vivo (68). This result is consistent with our finding that an RNP made up of a bacterial RPR + archaeal POP5•RPP30 is ∼50-fold weaker than the bacterial RNase P holoenzyme (50).…”

Section: Discussionmentioning

confidence: 99%

Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex

Chen¹,

Pulukkunat²,

Cho³

et al. 2010

Nucleic Acids Research

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RNase P catalyzes the Mg2+-dependent 5′-maturation of precursor tRNAs. Biochemical studies on the bacterial holoenzyme, composed of one catalytic RNase P RNA (RPR) and one RNase P protein (RPP), have helped understand the pleiotropic roles (including substrate/Mg2+ binding) by which a protein could facilitate RNA catalysis. As a model for uncovering the functional coordination among multiple proteins that aid an RNA catalyst, we use archaeal RNase P, which comprises one catalytic RPR and at least four RPPs. Exploiting our previous finding that these archaeal RPPs function as two binary RPP complexes (POP5•RPP30 and RPP21•RPP29), we prepared recombinant RPP pairs from three archaea and established interchangeability of subunits through homologous/heterologous assemblies. Our finding that archaeal POP5•RPP30 reconstituted with bacterial and organellar RPRs suggests functional overlap of this binary complex with the bacterial RPP and highlights their shared recognition of a phylogenetically-conserved RPR catalytic core, whose minimal attributes we further defined through deletion mutagenesis. Moreover, single-turnover kinetic studies revealed that while POP5•RPP30 is solely responsible for enhancing the RPR’s rate of precursor tRNA cleavage (by 60-fold), RPP21•RPP29 contributes to increased substrate affinity (by 16-fold). Collectively, these studies provide new perspectives on the functioning and evolution of an ancient, catalytic ribonucleoprotein.

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Function of heterologous and truncated RNase P proteins in Bacillus subtilis

Cited by 17 publications

References 48 publications

By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

By Any Other Name: Heterologous Replacement of the Escherichia coli RNase P Protein Subunit Has In Vivo Fitness Consequences

The putative RNase P motif in the DEAD box helicase Hera is dispensable for efficient interaction with RNA and helicase activity

Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex

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