Interaction of RNase P fromEscherichia coliwith pseudoknotted structures in viral RNAs

Abstract

In a previous study it was shown that RNase P from E. coli cleaves the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA in vitro (Guerrier-Takada et al. (1988) Cell, 53, 267-272). Cleavage takes place at the 3' side of the loop that crosses the deep groove of the pseudoknot structure present in the aminoacyl acceptor domain. In the present study fragments of TYMV RNA with mutations in the pseudoknot, generated by transcription in vitro, were tested for susceptibility to cleavage by RNase P. Changes in the specificity with respect to the site of cleavage and decreases in the rate of cleavage were observed with most of these substrates. The behaviour of various mutants in the reaction catalyzed by RNase P is in agreement with the present model of the TYMV RNA pseudoknot (Dumas et al. (1987), J. Biomol. Struct. Dyn. 263, 652-657). Base substitutions in the loop that crosses the shallow groove of the pseudoknot structure resulted, however, in an unexpected decrease in the rate of cleavage, probably due to conformational changes in the substrates. Studies on other tRNA-like structures revealed an important role in the reaction with RNase P for both the nucleotide at the 3' side of the loop that spans the deep groove and the nucleotide at position 4, which correspond to positions--1 and 73, respectively, in tRNA precursors.

Specific Cleavage of Hepatitis C Virus RNA Genome by Human RNase P

“…This proposed idea is further supported by the presence of a pseudoknot near the HCV IRES (43) cleavage site (a common element in tRNA-like structures including that which is known to interact with E. coli RNase P in the case of the tRNA-like motifs of plant viral RNAs) (42) (Fig. 8).…”

“…Subsequently, this and other plant viral RNAs were seen to be accessible to a battery of factors involved in other tRNA-related activities (including accessibility of bacterial RNase P) (37,41,42). Nevertheless, in vivo functional mimicry was not complete, since viral RNAs were not amino acid donors for protein synthesis but rather participated in virus replication.…”

Characterization of a cyanobacterial RNase P ribozyme recognition motif in the IRES of foot-and-mouth disease virus reveals a unique structural element

“…As shown in this study, the core structural element recognized by the cyanobacterial Rz 6803 resides in this region. Ribozyme recognition remarks the unity of the structural entity in the apical region of domain 3. tRNA-like structures recognized by RNase P enzymes in various viral RNAs are located in the vicinity of RNA pseudoknots that create complex structural organizations (Mans et al 1990). At present, it is not known if the apical domain threefolds in a pseudoknotted manner or if the GNRA-receptor interaction provides similar clues for Rz 6803 recognition.…”

“…This result was later expanded to pestivirus RNAs (Lyons and Robertson 2003). Similarly plant RNA viruses containing tRNA-like structures at the 39 end were recognized in vitro by RNase P (GuerrierTakada et al 1988;Mans et al 1990). In an independent approach, it has been described that IGR elements belonging to the Dicistroviridae family mimic the initiator tRNA during internal initiation (Spahn et al 2004;Schuler et al 2006) in a process independent of initiation factors and tRNA i (Wilson et al 2000;Jan and Sarnow 2002;Jan et al 2003;Pestova and Hellen 2003).…”

Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli

“…Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme that catalyzes the 59 maturation of all precursor transfer RNAs (ptRNAs)+ Precursors to Escherichia coli 4+5S RNA, tm RNA (10Sa RNA), Salmonella typhimurium his operon mRNA, and several bacteriophage encoded RNAs are also substrates for E. coli RNase P (Bothwell et al+, 1976a(Bothwell et al+, , 1976bGuerrier-Takada et al+, 1988;Mans et al+, 1990;Alifano et al+, 1994;Komine et al+, 1994;Hartmann et al+, 1995)+ E. coli RNase P is composed of a single catalytic RNA (M1 RNA, 377 nt) and a single protein cofactor (C5 protein, 119 amino acids) (Guerrier-Takada et al+, 1983)+ For the E. coli ptRNA substrates, important features for recognition by M1 RNA in vitro include the T stem/loop, the length of the acceptor stem, and the canonical 39 CCA terminus (McClain et al+, 1987;Kirsebom & Vioque, 1996)+ M1 RNA is believed to have the capacity to recognize its varied substrates through multiple binding modes (Guerrier-Takada et al+, 1989;Knap et al+, 1990;Guerrier-Takada & Altman, 1992)+ Such multiple binding modes could reflect an ability of this enzyme to assume more than one conformation on the pathway to an optimally active conformation that would ultimately lead to a cleavage event+ It has been proposed that the catalytic RNA component of Bacillus subtilis RNase P can assume two conformers to which a single substrate can bind, leading to productive enzyme-substrate complexes (Beebe & Fierke, 1994)+ Chemical probing experiments using Fe-EDTA and other methods have noted changes in M1 RNA structure following association with substrate, which could be indicative of an induced-fit type mechanism leading to the formation of a productive complex (Knap et al+, 1990;Guerrier-Takada & Altman, 1993;Ciesiolka et al+, 1994;Westhof et al+, 1996)+ The divalent metal ion and basic protein cofactors of RNase P may have key roles in modulating conformational changes of M1 RNA, both prior to and following substrate binding+ An increase in Mg 2ϩ concentration or the presence of C5 protein can alter the global architecture of M1 RNA (Westhof et al+, 1996)+ Further, deleterious mutations in M1 RNA can be rescued by high, nonphysiological salt concentration or C5 protein, indicative of their ability to assist M1 RNA in structure formation (Lumelsky & Altman, 1988;Gopalan et al+, 1994)+ C5 protein also increases the binding affinity of M1 RNA in a substrate-d...…”