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1.
Ribonuclease P (RNase P) is a catalytic ribonucleoprotein (RNP) essential for tRNA biosynthesis. In Escherichia coli, this RNP complex is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein. Using the sulfhydryl-specific reagent (1-oxyl-2,2,5, 5-tetramethyl-Delta3-pyrroline-3-methyl)methanethiosulfonate (MTSL), we have introduced a nitroxide spin label individually at six genetically engineered cysteine residues (i.e., positions 16, 21, 44, 54, 66, and 106) and the native cysteine residue (i.e., position 113) in C5 protein. The spin label covalently attached to any protein is sensitive to structural changes in its microenvironment. Therefore, we expected that if the spin label introduced at a particular position in C5 protein was present at the RNA-protein interface, the electron paramagnetic resonance (EPR) spectrum of the spin label would be altered upon binding of the spin-labeled C5 protein to M1 RNA. The EPR spectra observed with the various MTSL-modified mutant derivatives of C5 protein indicate that the spin label attached to the protein at positions 16, 44, 54, 66, and 113 is immobilized to varying degrees upon addition of M1 RNA but not in the presence of a catalytically inactive, deletion derivative of M1 RNA. In contrast, the spin label attached to position 21 displays an increased mobility upon binding to M1 RNA. The results from this EPR spectroscopy-based approach together with those from earlier studies identify residues in C5 protein which are proximal to M1 RNA in the RNase P holoenzyme complex. 相似文献
2.
R Brimacombe 《Biochimie》1991,73(7-8):927-936
Over the last two decades essentially three different approaches have been used to study the topography of RNA-protein interactions in the ribosome. These are: (a) the analysis of binding sites for individual ribosomal proteins or groups of proteins on the RNA; (b) the determination of protein footprint sites on the RNA by the application of higher order structure analytical techniques; and (c) the localisation of RNA-protein cross-link sites on the RNA. This article compares and contrasts the types of data that the three different approaches provide, and gives a brief and highly simplified summary of the results that have been obtained for both the 16S and 23S ribosomal RNA from E coli. 相似文献
3.
Importance of RNA-protein interactions in bacterial ribonuclease P structure and catalysis 总被引:1,自引:0,他引:1
Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) complex that catalyzes the metal-dependent maturation of the 5' end of precursor tRNAs (pre-tRNAs) in all organisms. RNase P is comprised of a catalytic RNA (P RNA), and at least one essential protein (P protein). Although P RNA is the catalytic subunit of the enzyme and is active in the absence of P protein under high salt concentrations in vitro, the protein is still required for enzyme activity in vivo. Therefore, the function of the P protein and how it interacts with both P RNA and pre-tRNA have been the focus of much ongoing research. RNA-protein interactions in RNase P serve a number of critical roles in the RNP including stabilizing the structure, and enhancing the affinity for substrates and metal ions. This review examines the role of RNA-protein interactions in bacterial RNase P from both structural and mechanistic perspectives. 相似文献
4.
Properties of purified ribonuclease P from Escherichia coli 总被引:12,自引:0,他引:12
The purified protein moiety of ribonuclease P (EC 3.1.26.5) from Escherichia coli, a single polypeptide of molecular weight approximately 17 500, has not catalytic activity by itself on several RNA substrates. However, when it is marked in vitro with an RNA species called M1 RNA, RNase P activity is reconstituted. The rate at which the purified RNase P cleaves any particular tRNA precursor molecule depends on the identity of that tRNA precursor. 相似文献
5.
Ribonucleoproteins (RNP) are involved in many essential processes in life. However, the roles of RNA and protein subunits in an RNP complex are often hard to dissect. In many RNP complexes, including the ribosome and the Group II introns, one main function of the protein subunits is to facilitate RNA folding. However, in other systems, the protein subunits may perform additional functions, and can affect the biological activities of the RNP complexes. In this review, we use ribonuclease P (RNase P) as an example to illustrate how the protein subunit of this RNP affects different aspects of catalysis. RNase P plays an essential role in the processing of the precursor to transfer RNA (pre-tRNA) and is found in all three domains of life. While every cell has an RNase P (ribonuclease P) enzyme, only the bacterial and some of the archaeal RNase P RNAs (RNA component of RNase P) are active in vitro in the absence of the RNase P protein. RNase P is a remarkable enzyme in the fact that it has a conserved catalytic core composed of RNA around which a diverse array of protein(s) interact to create the RNase P holoenzyme. This combination of highly conserved RNA and altered protein components is a puzzle that allows the dissection of the functional roles of protein subunits in these RNP complexes. 相似文献
6.
Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli 总被引:27,自引:0,他引:27
The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA. 相似文献
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8.
Six 50 S ribosomal subunit proteins, each unable to interact independently with the 23 S RNA, were shown to associate specifically with ribonucleoprotein complexes consisting of intact 23 S RNA, or fragments derived from it, and one or more RNA-binding proteins. In particular, L21 and L22 depend for attachment upon L20 and L24, respectively; L5, L10 and L11 interact individually with complexes containing L2 and L16; and one or both proteins of the mixture are stimulated to bind in the presence of L1, L3, L6, L13 and L23. Moreover, L14 alone was found to interact with a fragment from the 3′ end of the 23 S RNA, even though it cannot bind to 23 S RNA. By correlating the data reported here with the findings of others, it has been possible to formulate a partial in vitro assembly map of the Escherichia coli 50 S subunit encompassing both the 5 S and 23 S RNAs as well as 21 of the 34 subunit proteins. 相似文献
9.
Zhang XY Goemaere EL Seddiki N Célia H Gavioli M Cascales E Lloubes R 《The Journal of biological chemistry》2011,286(13):11756-11764
The tolQRAB-pal operon is conserved in Gram-negative genomes. The TolQRA proteins of Escherichia coli form an inner membrane complex in which TolQR uses the proton-motive force to regulate TolA conformation and the in vivo interaction of TolA C-terminal region with the outer membrane Pal lipoprotein. The stoichiometry of the TolQ, TolR, and TolA has been estimated and suggests that 4-6 TolQ molecules are associated in the complex, thus involving interactions between the transmembrane helices (TMHs) of TolQ, TolR, and TolA. It has been proposed that an ion channel forms at the interface between two TolQ and one TolR TMHs involving the TolR-Asp(23), TolQ-Thr(145), and TolQ-Thr(178) residues. To define the organization of the three TMHs of TolQ, we constructed epitope-tagged versions of TolQ. Immunodetection of in vivo and in vitro chemically cross-linked TolQ proteins showed that TolQ exists as multimers in the complex. To understand how TolQ multimerizes, we initiated a cysteine-scanning study. Results of single and tandem cysteine substitution suggest a dynamic model of helix interactions in which the hairpin formed by the two last TMHs of TolQ change conformation, whereas the first TMH of TolQ forms intramolecular interactions. 相似文献
10.
Shiying Wu Yu Chen Guanzhong Mao Stefan Trobro Marek Kwiatkowski Leif A. Kirsebom 《Nucleic acids research》2014,42(1):631-642
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. 相似文献
11.
A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with 32Pi at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P. 相似文献
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13.
Human tyrosine tRNA and fly alanine, histidine, and initiator methionine tRNAs are generally cleavable internally by bacterial ribonuclease P ribozyme. The unusual internal cleavage reaction of tRNA, called hyperprocessing, occurs when the cloverleaf structure of the tRNA molecule is denatured to form a double-hair-pin-like structure. The hyperprocessing reaction of these tRNAs requires magnesium ions. We analyzed details of this reaction using human tyrosine tRNA and Escherichia coli RNase P ribozyme. The usual processing reaction occurred efficiently with magnesium at 5 mM, but for the hyperprpocessing reaction, higher concentrations were needed. With such high concentrations, hyperprocessing cleaved both mature tRNA and tRNA precursor as substrates. When mature tRNA was the substrate, the apparent K(M) was almost the same as in the usual reaction, but k(cat) was smaller. These results indicated that the occurrence of hyperprocessing depends on the magnesium ion concentration, and suggested that magnesium ions contribute to the recognition of the shape of the substrate by bacterial RNase P enzymes. 相似文献
14.
Cleavage efficiencies of model substrates for ribonuclease P from Escherichia coli and Thermus thermophilus.
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We compared cleavage efficiencies of mono-molecular and bipartite model RNAs as substrates for RNase P RNAs (M1 RNAs) and holoenzymes from E. coli and Thermus thermophilus, an extreme thermophilic eubacterium. Acceptor stem and T arm of pre-tRNA substrates are essential recognition elements for both enzymes. Impairing coaxial stacking of acceptor and T stems and omitting the T loop led to reduced cleavage efficiencies. Small model substrates were less efficiently cleaved by M1 RNA and RNase P from T. thermophilus than by the corresponding E. coli activities. Competition kinetics and gel retardation studies showed that truncated tRNA substrates are less tightly bound by RNase P and M1 RNA from both bacteria. Our data further indicate that (pre-)tRNA interacts stronger with E. coli than T. thermophilus M1 RNA. Thus, low cleavage efficiencies of truncated model substrates by T. thermophilus RNase P or M1 RNA could be explained by a critical loss of important contact points between enzyme and substrate. In addition, acceptor stem--T arm substrates, composed of two synthetic RNA fragments, have been designed to mimic internal cleavage of any target RNA molecule available for base pairing. 相似文献
15.
Purification and properties of ribonuclease III from Escherichia coli 总被引:74,自引:0,他引:74
16.
17.
Subunit interactions in aspartate trancarbamylase from Escherichia coli studied using matrix-bound derivatives 总被引:1,自引:0,他引:1
W W Chan 《FEBS letters》1974,44(2):178-181
18.
Acid-soluble ribosomal ribonuclease of Escherichia coli 总被引:3,自引:0,他引:3
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20.
Samanta D Mukhopadhyay D Chowdhury S Ghosh J Pal S Basu A Bhattacharya A Das A Das D DasGupta C 《Journal of bacteriology》2008,190(9):3344-3352
The peptidyl transferase center, present in domain V of 23S rRNA of eubacteria and large rRNA of plants and animals, can act as a general protein folding modulator. Here we show that a few specific nucleotides in Escherichia coli domain V RNA bind to unfolded proteins and, as shown previously, bring the trapped proteins to a folding-competent state before releasing them. These nucleotides are the same for the proteins studied so far: bovine carbonic anhydrase, lactate dehydrogenase, malate dehydrogenase, and chicken egg white lysozyme. The amino acids that interact with these nucleotides are also found to be specific in the two cases tested: bovine carbonic anhydrase and lysozyme. They are either neutral or positively charged and are present in random coils on the surface of the crystal structure of both the proteins. In fact, two of these amino acid-nucleotide pairs are identical in the two cases. How these features might help the process of protein folding is discussed. 相似文献