| 1. |
- Kirsebom, Leif A, et al.
(författare)
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Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors
- 1988
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 204:4, s. 879-888
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.
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| 2. |
- Kirsebom, Leif A, et al.
(författare)
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Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates
- 1989
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 207:4, s. 837-840
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The reaction of wild-type and two mutant derivatives of RNase P have been examined with wild-type and mutant substrates. We show that a mutant derivative of tRNA(Tyr)Su3, tRNA(Tyr)Su3A15, in which the G15.C48(57) base-pair essential for folding of the tRNA moiety is altered, is a temperature-sensitive suppressor in vivo. The precursor to tRNA(Tyr)Su3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher Km compared to the precursor to tRNA(Tyr)Su3. The precursor to tRNA(Tyr)Su3A2, another temperature-sensitive suppressor in vivo in which the G2.C71(80) base-pair in the acceptor stem is changed to A2.C71(80), behaves like the precursor to tRNA(Tyr)Su3 in vitro; that is, it is not cleaved in a temperature-sensitive manner. Therefore, there are at least two ways in which a suppressor tRNA can acquire a temperature-sensitive phenotype in vivo. One of the mutant derivatives of RNase P we have tested, rnpA49, which affects the protein cofactor of the enzyme, has a decreased kcat compared to wild-type, which can explain its phenotype in vivo.
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| 3. |
- Brännvall, Mathias, et al.
(författare)
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Evidence for induced fit in bacterial RNase P RNA-mediated cleavage
- 2007
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 372:5, s. 1149-1164
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Tidskriftsartikel (refereegranskat)abstract
- RNase P with its catalytic RNA subunit is involved in the processing of a number of RNA precursors with different structures. However, precursor tRNAs are the most abundant substrates for RNase P. Available data suggest that a tRNA is folded into its characteristic structure already at the precursor state and that RNase P recognizes this structure. The tRNA D-/T-loop domain (TSL-region) is suggested to interact with the specificity domain of RNase P RNA while residues in the catalytic domain interact with the cleavage site. Here, we have studied the consequences of a productive interaction between the TSL-region and its binding site (TBS) in the specificity domain using tRNA precursors and various hairpin-loop model substrates. The different substrates were analyzed with respect to cleavage site recognition, ground-state binding, cleavage as a function of the concentration of Mg2+ and the rate of cleavage under conditions where chemistry is suggested to be rate limiting using wild-type Escherichia coli RNase P RNA, M1 RNA, and M1 RNA variants with structural changes in the TBS-region. On the basis of our data, we conclude that a productive TSL/TBS interaction results in a conformational change in the M1 RNA substrate complex that has an effect on catalysis. Moreover, it is likely that this conformational change comprises positioning of chemical groups (and Mg2+) at and in the vicinity of the cleavage site. Hence, our findings are consistent with an induced-fit mechanism in RNase P RNA-mediated cleavage.
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| 4. |
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| 5. |
- Pettersson, B. M. Fredrik, 1974-, et al.
(författare)
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The Presence of a C− 1/G+ 73 Pair in a tRNA Precursor Influences Processing and Expression In Vivo
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 381:5, s. 1089-1097
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Tidskriftsartikel (refereegranskat)abstract
- To understand whether 5′ and 3′ structural elements of the region corresponding to the mature tRNA affect the expression of the tRNA, we examined several bacterial genomes for tRNA genes where the expression might be potentially affected by structural elements located outside of the mature tRNA. In Pseudomonas aeruginosa, our analysis suggested that the tRNATrp is transcribed together with a putative stem–loop structure followed by a uridine tract immediately downstream of the tRNA region. This structural element, resembling a Rho-independent transcription terminator, might therefore influence the expression and processing of tRNATrp. Moreover, the secondary structure suggested that the discriminator base in the tRNATrp precursor can pair with either the C at position − 1, the 3′ terminal residue in the 5′ leader, or the C immediately 5′ of the uridine tract of the putative Rho-independent transcription terminator. Here, we present in vivo data demonstrating the importance of residue − 1 and the positioning of the putative transcription terminator for the expression of correctly 5′ processed P. aeruginosa tRNATrp in Escherichia coli. Interestingly, we also detected a difference in the appearance of correctly 5′ processed P. aeruginosa tRNATrp in E. coli compared to the situation in P. aeruginosa.
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| 6. |
- Wu, Shiying, 1978-, et al.
(författare)
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Cleavage mediated by the catalytic domain of bacterial RNase P RNA
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 422:2, s. 204-214
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Tidskriftsartikel (refereegranskat)abstract
- As for other RNA molecules RNase P RNA (RPR) is composed of domains and these have different functions. Here we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model hairpin loop substrates. Compared to full-size Eco RPR the rate of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13000-fold. We provide data that the magnitude of reduction in rate is substrate dependent and that the structural architecture of the -1/+73 plays a significant role where a C-1/G+73 pair had the most dramatic effect on the rate. Substitution of A248 (E. coli numbering), which is positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold rate improvement while strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, while deleting the S-domain gave a reduction in the rate it resulted in a less erroneous RPR with respect to cleavage site selection. These data will be dicussed in view of our current understanding of the coupling between the distal interaction between the S-domain and events at the active site and in an evolutionary perspective.
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| 7. |
- Wu, Shiying, et al.
(författare)
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Functional Coupling between a Distal Interaction and the Cleavage Site in Bacterial RNase-P-RNA-Mediated Cleavage
- 2011
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 411:2, s. 384-396
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial RNase P consists of one protein and one RNA [RNase P RNA (RPR)]. RPR can process tRNA precursors correctly in the absence of the protein. Here we have used model hairpin loop substrates corresponding to the acceptor, T-stem, and T-loop of a precursor tRNA to study the importance of the T-loop structure in RPR-alone reaction. T-stem/loop (TSL) interacts with a region in RPR [TSL binding site (TBS)], forming TSL/TBS interaction. Altering the T-loop structure affects both cleavage site selection and rate of cleavage at the correct site +1 and at the alternative site -1. The magnitude of variation depended on the structures of the T-loop and the TBS region, with as much as a 150-fold reduction in the rate of cleavage at +1. Interestingly, for one T-loop structure mutant, no difference in the rate at -1 was detected compared to cleavage of the substrate with an unchanged T-loop, indicating that, in this case, the altered T-loop structure primarily influences events required for efficient cleavage at the correct site +1. We also provide data supporting a functional link between a productive TSL/TBS interaction and events at the cleavage site. Collectively, our findings emphasize the interplay between separate regions upon formation of a productive RPR substrate that leads to efficient and accurate cleavage. These new data provide support for an induced-fit mechanism in bacterial RPR-mediated cleavage at the correct site +1.
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