Long-Range Structure in Ribonuclease P RNA

Haas, Elizabeth S.; Morse, Daniel P.; Brown, James W.; Schmidt, Francis J.; Pace, Norman R.

doi:10.1126/science.1719634

Cited by 93 publications

(79 citation statements)

References 21 publications

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“…The energetics of the P1-P6 helix also may play a role, because the U6C paused intermediate structure may not be labile enough to allow for efficient folding once the downstream portions are transcribed. We could not readily test this possibility with compensatory mutations for residue G83, because it is located in the P6 pseudoknot crucial for the native structure of RNase P RNA (24,25). Mutating G83 requires additional mutations elsewhere to restore the native structure, and folding results from such complex mutants would have been difficult to interpret.…”

Section: Resultsmentioning

confidence: 99%

Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures

Wong¹,

Sosnick

Pan³

2007

Proc. Natl. Acad. Sci. U.S.A.

119

118

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RNA folding in the cell occurs during transcription. Expedient RNA folding must avoid the formation of undesirable structures as the nascent RNA emerges from the RNA polymerase. We show that efficient folding during transcription of three conserved noncoding RNAs from Escherichia coli, RNase P RNA, signal-recognition particle RNA, and tmRNA is facilitated by their cognate polymerase pausing at specific locations. These pause sites are located between the upstream and downstream portions of all of the native long-range helices in these noncoding RNAs. In the paused complexes, the nascent RNAs form labile structures that sequester these upstream portions in a manner to possibly guide folding. Both the pause sites and the secondary structure of the nonnative portions of the paused complexes are phylogenetically conserved among ␥-proteobacteria. We propose that specific pausing-induced structural formation is a general strategy to facilitate the folding of long-range helices. This polymerase-based mechanism may result in portions of noncoding RNA sequences being evolutionarily conserved for efficient folding during transcription.RNase P ͉ tmRNA ͉ long-range helix B ecause of the degeneracy and stability of base-pairing, RNA has a high propensity to form long-lived undesirable intermediates along their folding pathways (1-3). Particularly challenging for RNA folding during transcription is the formation of duplexes composed of two strands located far apart in sequence. In the time before the transcription of the downstream strand, the upstream strand can form stable yet nonnative base pairs with other regions, which could hinder folding. This undesirable possibility can be circumvented by sequestering the upstream strand in specific but labile structures. Such structures can guide the formation of the native long-range helices by avoiding the formation of long-lived unproductive species.When compared with Mg 2ϩ -induced refolding of full-length RNAs, two obvious properties that influence RNA folding during transcription are the 5Ј to 3Ј polarity of RNA synthesis and the transcriptional speed of the RNA polymerase (4-8). In addition, bacterial RNA polymerases pause during transcriptional elongation (9, 10). The location and/or duration of the pause sites can alter folding (7, 11). A previous work on the folding during transcription of a circularly permuted version of the Bacillus subtilis RNase P RNA demonstrated that pausing at a specific site alters its folding during transcription by Escherichia coli RNA polymerase (11). Only the pause at this site was required for the accelerated folding of one of the two domains of P RNA; varying the transcriptional speed had no effect. Pausing at this site altered the nascent RNA so that an undesirable structure involving a region downstream to the pause site was avoided.Although the above result demonstrated that pausing can influence folding, it did not address the question of biological relevancy, because (i) circularly permuted RNA transcripts are unnatural, having changed t...

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Section: Resultsmentioning

confidence: 99%

Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures

Wong¹,

Sosnick

Pan³

2007

Proc. Natl. Acad. Sci. U.S.A.

119

118

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“…If transcription begins in multiple locations, a discrete transcript would not be seen. All RNase P RNAs contain two short regions of conserved sequence similarity presumed to participate in the formation of a pseudoknot thought to be necessary for activity (20). The two altered genes used in this study were created by site directed mutagenesis targeted to the 5' most of these conserved sequences.…”

Section: Discussionmentioning

confidence: 99%

“…Figure 2A shows the secondary structure interaction which occurs between the 5' and 3' regions of E.coli RNase P RNA (20) while Figure 2B shows the predicted interaction in S.cerevisiae. Both altered genes used in this study contained changes in the 5' conserved sequence and were created by in vitro mutagenesis as described in Materials and Methods.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Successful transformation of yeast mitochondria withRPM1: an approach forin vivostudies of mitochondrial RNase P RNA structure, function and biosynthesis

et al. 1995

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Mitochondrial RNase P RNA (Rpmlr) is coded by the RPM1 gene of mitochondrial DNA in many yeasts. As an initial step to developing a genetic approach to the structure and biogenesis of yeast mitochondrial RNase P, biolistic transformation has been used to introduce wild type and altered RPM1 genes into strains containing no mitochondrial DNA. The introduced wild type gene does support RNase P activity demonstrating that pre existing RNase P activity is not necessary for the biosynthesis of the enzyme. Mutations introduced into RPM1 in vitro result in reduced accumulation of mature tRNA and in an alteration of the processing of Rpml r in vivo.

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“…A's 65-67 are part of the pseudoknot containing P4 (Haas et al 1991), and are important for catalytic activity (Kazantsev and Pace 1998). A's 98 and 99 in L8 are predicted to be involved in a tertiary interaction with P4 (Massire et al 1998).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA

Childs

Poole²,

Turner³

2003

RNA

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Oligonucleotide directed misfolding of RNA (ODMiR) uses short oligonucleotides to inhibit RNA function by exploiting the ability of RNA to fold into different structures with similar free energies. It is shown that the 2 -O-methyl oligonucleotide, m(CAGCCUACCCGG), can trap Escherichia coli RNase P RNA (M1 RNA) in a nonfunctional structure in a transcription mixture containing RNase P protein (C5 protein). At about 200 nM, the 12-mer thus inhibits 50% of pre-tRNA processing by RNase P. Roughly 10-fold more 12-mer is required to inhibit RNase P containing full-length, renatured RNase P RNA. Diethyl pyrocarbonate modification in the presence of 12-mer reveals increased modification of sites in and interacting with P4, suggesting a structural rearrangement of a large pseudoknot important for catalytic activity. Thus, the ODMiR method can be applied to RNAs even when folding is facilitated by a cognate protein.

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Long-Range Structure in Ribonuclease P RNA

Cited by 93 publications

References 21 publications

Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures

Folding of noncoding RNAs during transcription facilitated by pausing-induced nonnative structures

Successful transformation of yeast mitochondria withRPM1: an approach forin vivostudies of mitochondrial RNase P RNA structure, function and biosynthesis

Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA

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