Studies on RNase T1 mutants affecting enzyme catalysis

Grunert, Hans‐Peter; Zouni, Athina; Beineke, Marc; Quaas, Rainer; Georgalis, Yannis; Saenger, Wolfram; Hahn, Ulrich

doi:10.1111/j.1432-1033.1991.tb15900.x

Cited by 40 publications

(16 citation statements)

References 37 publications

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“…42, No. 6, 20031427 His40 of RNase T1 which has been shown to be not indispensable (31) and capable of acting as a base in place of Glu58, when replaced, resulted not overimposed with any putative residues of Sso7d. The comparison between Sso7d and some RNase T1 mutants is even more interesting since significant activity in the double mutant His92,His40/Asp has been demonstrated (32).…”

Section: Sso7d Catalytic Residuesmentioning

confidence: 98%

Investigations of Sso7d Catalytic Residues by NMR Titration Shifts and Electrostatic Calculations

et al. 2003

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Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.

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Section: Sso7d Catalytic Residuesmentioning

confidence: 98%

Investigations of Sso7d Catalytic Residues by NMR Titration Shifts and Electrostatic Calculations

et al. 2003

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“…Recombinant RNase T1 in the isoenzyme form (with Lys25 instead of Gln25 in the mother enzyme) was expressed in Escherichia coli and isolated as described (Grunert et al, 1991;Quaas et al, 1988a, b). A solution containing 1% RNase T1, 1 mM Gp[S]U, 20 mM sodium acetate, 2 mM calcium acetate, pH 4.0, was equilibrated against 50% 2-methyl-2,4-pentanediol using the sitting-drop vapor-diffusion method.…”

Section: Experimental Procedures Crystallographymentioning

confidence: 99%

The complex between ribonuclease T1 and 3′GMP suggests geometry of enzymic reaction path

Heydenreich

Koellner

Choe

et al. 1993

European Journal of Biochemistry

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The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 NEH' activates 02'-H, (d) Glu58 carboxylate acts as base and His92 NEH' as acid in a general acid-base catalysis.The crystals have the monoclinic space group P2,, a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, p = 90.62" with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11338 data r l a ( F , ) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle E in trans. The 02'-H group is activated by accepting and donating hydrogen bonds from His40 NEH' and to Glu58 081; the phosphate is hydrogen bonded to Glu58 O E~H , Arg77 NEH' and Ny2H', Tyr38 OyH, His92 NEH'. The conformation of ribose phosphate is such that 02' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 NEH' ; we infer from a model building study that this bond is equivalent to the scissile P-05' in a substrate GpN.Ribonuclease T1 (RNase T1) from the fungus Aspergillus oryzae is the key representative of a family of homologous microbial ribonucleases. It has been studied extensively in terms of enzymology (Takahashi and Moore, 1982) and molecular structure (Pace et al., 1991). The chemical function of RNase TI is to cleave the RNA phosphodiester bond specifically at the 3' side of guanosine. The reaction occurs in two steps, first a transesterification to yield oligonucleotides with terminal guanosine 2',3'-monophosphate (2',3'c GPM; Scheme I), and second the hydrolysis of this reaction intermediate to guanosine 3'-monophosphate (3'GMP) (Saenger, 1991).With the present study, we attempted to determine the structure of an enzyme-substrate complex by cocrystallizing RNase T1 with the substrate analogue guanosine-(3')thiophospho(5')uridine (Gp[S]U). In the synthesis of this analogue, two diastereoisomers are obtained with the thiophosphate group in R, and S, configurations (Eckstein et al., 1972). Since the S, isomer is not or only very slowly cleaved by RNase T1, we hoped that it could be cocrystallized with the enzyme to form a non-productive complex.

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“…The E105A mutation abolishes RegB toxicity, and E19A and E19V lower it. Previous mutagenesis experiments with other ribonucleases (including YoeB) showed that mutants of the general base conserved residual activity (30,32,33 19 and His 48 C ␤ is slightly too long. This could be related to the observation that RegB is catalytically a "very poor" enzyme and could suggest that the formation of a productive enzyme-substrate complex involves, after a first rapid binding step, an infrequent rearrangement of the protein structure.…”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Studies of RegB, a New Member of a Family of Sequence-specific Ribonucleases Involved in mRNA Inactivation on the Ribosome

Odaert

Saïda

Aliprandi

et al. 2007

Journal of Biological Chemistry

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The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.Control of messenger RNA decay is a key factor in the regulation of gene expression. Indeed, the expression level of a protein is strongly influenced by the concentration of its mRNA, which, in turn, depends on its synthesis and degradation rates (1). Perturbation of mRNA stability can lead to dramatic effects on cell physiology and in some cases can induce cancer (2). However, the way the cell controls the lifetime of its mRNA is still a poorly understood aspect in the biology of both prokaryotic and eukaryotic organisms. In Escherichia coli, it is generally agreed that the initiation of mRNA degradation is induced by an endoribonucleolytic cleavage mediated by ribonucleases E and G with the occasional involvement of RNases III and P (3, 4). These ribonucleases all generate 5Ј-phosphate RNA fragments (RNases E, G, and III are metallo-enzymes; RNase P is a ribozyme), have broad specificity, and are involved in other cellular functions. RNases E and G, for example, participate in the maturation of 9 S and 16 S rRNA (5), and RNase E is necessary for tRNA processing (6).Ribonuclease RegB is radically different from the ribonucleases described above. RegB is involved in the control of the bacteriophage T4 multiplication cycle. It is produced in the early stage of viral infection and facilitates the transition between the early and middle phases of the viral cycle by inactivating early mRNAs (7). RegB cleaves its substrates with high specificity in the middle of the GGAG motif found in the ribosome-binding site of most prokaryotic mRNAs (Shine-Dalgarno sequence). Strikingly, it remains active throughout the entire lytic cycle but does not act on middle or late mRNA (8). In contrast with RNases E, G, III, and P, RegB is only involved in this task and is certainly one of the most specific ribonucleases described to date. It only cleaves GGAG, or to a lesser extent and with reduced efficiency, GGAU sequences (7, 9). Moreover, it does not cleave all such sequences: those present in coding sequences and in middle or late T4 mRNAs are ...

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Studies on RNase T1 mutants affecting enzyme catalysis

Cited by 40 publications

References 37 publications

Investigations of Sso7d Catalytic Residues by NMR Titration Shifts and Electrostatic Calculations

Investigations of Sso7d Catalytic Residues by NMR Titration Shifts and Electrostatic Calculations

The complex between ribonuclease T1 and 3′GMP suggests geometry of enzymic reaction path

Structural and Functional Studies of RegB, a New Member of a Family of Sequence-specific Ribonucleases Involved in mRNA Inactivation on the Ribosome

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