| 1. |
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| 2. |
- Herrmann, Björn, et al.
(författare)
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Differentiation and Phylogenetic Relationships in Mycobacterium spp with Special Reference to the RNase P RNA Gene rnpB
- 2014
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Ingår i: Current Microbiology. - : Springer Science and Business Media LLC. - 0343-8651 .- 1432-0991. ; 69:5, s. 634-639
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Tidskriftsartikel (refereegranskat)abstract
- The rnpB gene encodes for the RNA subunit of the catalytic ribonuclease RNase P and is present in all bacteria and has both conserved and highly variable sequence regions. Determination of rnpB in 35 Mycobacterium spp. showed species specific sequences for all species except the Mycobacterium tuberculosis complex (four species). High sequence variation was seen in the P3, P15 and P19 regions of suggested secondary structures of the corresponding RNase P RNA molecules. Phylogenetic analysis showed that rnpB gave similar tree topologies as 16S rRNA and hsp65 genes. A combined analysis of the three genes increased the number of nodes with significant support from 10 to 19. The results indicate that rnpB is useful for phylogenetic studies and is a possible target for identification and detection of Mycobacterium spp.
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| 3. |
- Kikovska, Ema, et al.
(författare)
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Cleavage mediated by the P15 domain of bacterial RNase P RNA
- 2012
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:5, s. 2224-2233
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Tidskriftsartikel (refereegranskat)abstract
- Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3'-end of tRNA precursors resulting in the formation of the RCCA-RNase P RNA interaction (interacting residues underlined) in the bacterial RPR-substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg2+ at the cleavage site. Here we show that small model-RNA molecules (similar to 30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for 'RNA-mediated' cleavage of RNA.
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| 4. |
- Kirsebom, Leif A., et al.
(författare)
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Pb2+-Induced Cleavage of RNA
- 2014. - 2
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Ingår i: Handbook of RNA Biochemistry. - Weinheim, Germany : Wiley-Blackwell. - 9783527647064 - 9783527327645 ; , s. 269-284
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Bokkapitel (refereegranskat)
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| 5. |
- Kirsebom, Leif A., et al.
(författare)
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Pleiomorphism in Mycobacterium
- 2012
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Ingår i: Advances in Applied Microbiology, Vol 80. - : ELSEVIER ACADEMIC PRESS INC. - 9780123943811 ; , s. 81-112
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Bokkapitel (refereegranskat)abstract
- Morphological variants in mycobacterial cultures under different growth conditions, including aging of the culture, have been shown to include fibrous aggregates, biofilms, coccoids, and spores. Here we discuss the diversity in shape and size changes demonstrated by bacterial cells with special reference to pleiomorphism observed in Mycobacterium spp. in response to nutritional and other environmental stresses. Inherent asymmetry in cell division and compartmentalization of cell interior under different growth conditions might contribute toward the observed pleiomorphism in mycobacteria. The regulatory genes comprising the bacterial signaling pathway responsible for initiating morphogenesis are speculated upon from bioinformatic identifications of genes for known sensors, kinases, and phosphatases existing in mycobacterial genomes as well as on the basis of what is known in other bacteria.
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| 6. |
- Kirsebom, Leif A
(författare)
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Roles of metal ions in RNase P catalysis
- 2010
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Ingår i: Ribonuclease P. - New York : Springer-Verlag New York. - 9781441911421 - 9781441911414 ; , s. 113-134
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- As for other RNA, RNase P with its catalytic RNA subunit requires metal(II)-ions for function. Approximately 100 metal(II)-ions bind to the 400 residues long RNA and several to the precursor substrate, e.g., roughly 25–30 to a tRNA precursor substrate. To understand the function and the reaction catalyzed by RNase P an important task is to identify and characterize metal(II)-ions or metal binding sites that contribute to folding of the RNAs, interaction with the protein subunit(s), substrate binding and chemistry of cleavage. Over the years, different methods have been explored to extract information about how, were and when metal(II)-ions bind to RNA. In this chapter, I will discuss our current understanding of RNase P and metal(II)-ions and how this knowledge can be utilized to search for new candidate drugs referred to as metal mimics.
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| 7. |
- Lai, Lien B., et al.
(författare)
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Unexpected diversity of RNase P, an ancient tRNA processing enzyme : Challenges and prospects
- 2010
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 584:2, s. 287-296
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Forskningsöversikt (refereegranskat)abstract
- For an enzyme functioning predominantly in a seemingly housekeeping role of 50 tRNA maturation, RNase P displays a remarkable diversity in subunit make-up across the three domains of life. Despite the protein complexity of this ribonucleoprotein enzyme increasing dramatically from bacteria to eukarya, the catalytic function rests with the RNA subunit during evolution. However, the recent demonstration of a protein-only human mitochondrial RNase P has added further intrigue to the compositional variability of this enzyme. In this review, we discuss some possible reasons underlying the structural diversity of the active sites, and use them as thematic bases for elaborating new directions to understand how functional variations might have contributed to the complex evolution of RNase P.
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| 8. |
- Pettersson, B M Fredrik, et al.
(författare)
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Draft Genome Sequence of Saccharopolyspora rectivirgula.
- 2014
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Ingår i: Genome Announcements. - 2169-8287. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- We have sequenced the genome of Saccharopolyspora rectivirgula, the causative agent of farmer's lung disease. The draft genome consists of 182 contigs totaling 3,977,051 bp, with a GC content of 68.9%.
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| 9. |
- Pettersson, B. M. Fredrik, et al.
(författare)
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Identification and expression of stressosomal proteins in Mycobacterium marinum under various growth and stress conditions
- 2013
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 0378-1097 .- 1574-6968. ; 342:2, s. 98-105
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Tidskriftsartikel (refereegranskat)abstract
- Like other bacteria, Mycobacterium spp. have developed different strategies in response to environmental changes such as nutrient limitations and other different stress situations. We have identified candidate genes (rsb genes) from Mycobacterium marinum involved in the regulation of the activity of the alternative sigma factor, sigma F. This is a homolog of the master regulator of general stress response, sigma B, and the sporulation-specific sigma factor, sigma F, in Bacillus subtilis. The organization of these genes in M.marinum and B.subtilis is similar. Transcriptome and qRT-PCR data show that these genes are indeed expressed in M.marinum and that the levels of expression vary with growth phase and exposure to stress. In particular, cold stress caused a significant rise in the expression of all identified rsb and sigF genes. We discuss these data in relation to what is currently known for other Mycobacterium spp.
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| 10. |
- Pettersson, B M Fredrik, et al.
(författare)
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tRNA accumulation and suppression of the bldA phenotype during development in Streptomyces coelicolor
- 2011
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 79:6, s. 1602-1614
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Tidskriftsartikel (refereegranskat)abstract
- Streptomyces coelicolor undergoes distinct morphological changes as it grows on solid media where spores differentiate into vegetative and aerial mycelium that is followed by the production of spores. Deletion of bldA, encoding the rare tRNA(Leu) UAA, blocks development at the stage of vegetative mycelium formation. From previous data it appears that tRNA(Leu) UAA accumulates relatively late during growth while two other tRNAs do not. Here, we studied the expression of 17 different tRNAs including bldA tRNA, and the RNA subunit of the tRNA processing endoribonuclease RNase P. Our results showed that all selected tRNAs and RNase P RNA increased with time during development. However, accumulation of bldA tRNA and another rare tRNA(Leu) isoacceptor started at an earlier stage compared with the other tRNAs. We also introduced the bldA tRNA anticodon (UAA) into other tRNAs and introduced these into a bldA deletion strain. In particular, one such mutant tRNA derived from the tRNA(Leu) CAA isoacceptor suppressed the bldA phenotype. Thus, the bldA tRNA scaffold is not critical for function as a regulator of S. coelicolor cell differentiation. Further substitution experiments, in which the 5'- and 3'-flanking regions of the suppressor tRNA were changed, indicated that these regions were important for the suppression.
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| 11. |
- Sinapah, Sylvie, et al.
(författare)
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Cleavage of model substrates by archaeal RNase P : role of protein cofactors in cleavage-site selection
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 39:3, s. 1105-1116
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Tidskriftsartikel (refereegranskat)abstract
- RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.
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| 12. |
- Singh, Bhupender, et al.
(författare)
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Asymmetric growth and division in Mycobacterium spp. : compensatory mechanisms for non-medial septa
- 2013
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 88:1, s. 64-76
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacterium spp., rod-shaped cells belonging to the phylum Actinomycetes, lack the Min- and Noc/Slm systems responsible for preventing the placement of division sites at the poles or over the nucleoids to ensure septal assembly at mid-cell. We show that the position for establishment of the FtsZ-ring in exponentially growing Mycobacterium marinum and Mycobacterium smegmatis cells is nearly random, and that the cells often divide non-medially, producing two unequal but viable daughters. Septal sites and cellular growth disclosed by staining with the membrane-specific dye FM4-64 and fluorescent antibiotic vancomycin (FL-Vanco), respectively, showed that many division sites were off-centre, often over the nucleoids, and that apical cell growth was frequently unequal at the two poles. DNA transfer through the division septum was detected, and translocation activity was supported by the presence of a putative mycobacterial DNA translocase (MSMEG2690) at the majority of the division sites. Time-lapse imaging of single live cells through several generations confirmed both acentric division site placement and unequal polar growth in mycobacteria. Our evidence suggests that post-septal DNA transport and unequal polar growth may compensate for the non-medial division site placement in Mycobacterium spp.
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| 13. |
- Singh, Bhupender
(författare)
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Dynamic Organization of Molecular Machines in Bacteria
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism.In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phaseBacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase.In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.
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| 14. |
- Singh, Bhupender, et al.
(författare)
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Growth, cell division and sporulation in mycobacteria
- 2010
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Ingår i: Antonie van Leeuwenhoek. International Journal of General and Molecular Microbiology. - : Springer Science and Business Media LLC. - 0003-6072 .- 1572-9699. ; 98:2, s. 165-177
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria have the ability to adapt to different growth conditions and to survive in various environments. They have also the capacity to enter into dormant states and some bacteria form spores when exposed to stresses such as starvation and oxygen deprivation. Sporulation has been demonstrated in a number of different bacteria but Mycobacterium spp. have been considered to be non-sporulating bacteria. We recently provided evidence that Mycobacterium marinum and likely also Mycobacterium bovis bacillus Calmette-Gu,rin can form spores. Mycobacterial spores were detected in old cultures and our findings suggest that sporulation might be an adaptation of lifestyle for mycobacteria under stress. Here we will discuss our current understanding of growth, cell division, and sporulation in mycobacteria.
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| 15. |
- 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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| 16. |
- Wu, Shiying, 1978-
(författare)
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Distal to Proximal—Functional Coupling in RNase P RNA-mediated Catalysis
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RNase P is a ubiquitous ribonuclease responsible for removing the 5’ leader of tRNA precursor. Bacterial RNase P contains one RNA (RPR) and one protein (RPP) subunit. However, the number of protein variants depends on the origin. The RNA subunit is the catalytic subunit that in vitro cleaves its substrate with and without the protein subunit. Therefore RNase P is a ribozyme. However, the protein subunit is indispensable in vivo. The objective of this thesis was to understand the mechanism of and substrate interaction in RPR-mediated cleavage, in particular the contributions of the two domains of RPR and the roles of the base at the -1 residue in the substrate. As model systems I have used bacterial (Eco) and archaeal (Pfu) RPRs. The TSL (T-stem-loop) region of a tRNA precursor and the TBS (TSL-binding site) in the RPR S-domain interact upon RPR-substrate complex conformation. A productive TSL/TBS-interaction affects events at the cleavage site by influencing the positioning of chemical groups and/ or Mg2+ such that efficient and correct cleavage occurs consistent with an induced fit mechanism. With respect to events at the cleavage site, my data show that the identity of the residue immediately upstream the 5’ of the cleavage site (at -1) plays a significant role for efficient and accurate cleavage although its presence is not essential. My data also show that the RPR C-domain can cleave without the S-domain. However, the presence of the S-domain increases the efficiency of cleavage but lowers the accuracy. The structure of the S-domain of Pfu RPR differs from that of Eco RPR and my data suggest that the Pfu S-domain does not affect the accuracy in the same way as for Eco RPR. It also appears that the proteins that bind to the Pfu S-domain play a role in formation of a productive TSL/TBS-interaction. It is therefore possible that the proteins of Pfu RNase P have evolved to take over the role of the S-domain with respect to the interaction with the TSL-region of the substrate.
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| 17. |
- 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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| 18. |
- Wu, Shiying, et al.
(författare)
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Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates
- 2014
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Ingår i: Nucleic Acids Research. - 0305-1048 .- 1362-4962. ; 42:1, s. 631-642
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Tidskriftsartikel (refereegranskat)abstract
- We have used model substrates carrying modified nucleotides at the site immediately 5' of the canonical RNase P cleavage site, the -1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at -1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at -1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at -1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the -1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.
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| 19. |
- Wu, Shiying, et al.
(författare)
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Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:1, s. 631-642
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Tidskriftsartikel (refereegranskat)abstract
- We have used model substrates carrying modified nucleotides at the site immediately 5' of the canonical RNase P cleavage site, the -1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at -1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at -1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at -1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the -1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H2O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.
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