Wild-type Substrate Research Articles

The detection of hammerhead ribozyme cleavage by RT-PCR methods.

AbstractThere are several methods available for the direct and indirect detection of hammerhead ribozyme-mediated cleavage of substrate RNAs in vivo. The indirect methods include the use of antisense and/or mutant ribozyme constructs to differentiate between inactivation by cleavage and inactivation by other means. In addition, comparison between a wild-type substrate and a mutant substrate, in which the target triplet has been altered to be a non-cleavable sequence, can provide indirect evidence for in vivo cleavage. The direct approaches (listed in increasing levels of sensitivity) include RNA analysis by Northern blot, RNase protection assays, and reverse transcriptase-PCR (RT-PCR) (but see also Chapters 33 and 34). To date there has only been one report of the direct analysis of cleavage products by Northern blot (1), and one group have successfully applied RNase protection assays (2–4).KeywordsHammerhead Ribozyme CleavageTarget TripletWild-type SubstrateRNA SubstrateMutant SubstrateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.

Influence of substrate structure on cleavage by hammerhead ribozyme

We compared the cleavage by a hammerhead ribozyme of a wild-type precursor tRNA (pre-tRNA 3 leu) and a structurally altered mutant form. We also analyzed the cleavage reactions of these tRNAs catalyzed by a ribozyme variant that was designed to complement the mutant precursor tRNA. Kinetic analyses reveal that the k cat values are nearly the same for the wild-type and the mutant substrate RNAs. However, the K m values differ considerably, being higher for the wild-type substrate. Thus, the formation of the ribozyme-substrate complex, but not the chemical cleavage step, is affected by these changes. Time course studies were performed, at different temperatures, to estimate the efficiency of the cleavage reactions and the effect of temperature. The cleavage of mutant precursor tRNA is generally faster than the wild-type at all temperatures analyzed. These results suggest that substrate structures can limit ribozyme efficiency, presumably by hindering the hybridization step.

Furilisin: a variant of subtilisin BPN' engineered for cleaving tribasic substrates.

The serine protease, subtilisin BPN', was engineered to cleave proteins after tribasic sequences in a manner that resembles the substrate specificity of furin, one of the mammalian subtilisin homologs that processes prohormones. As a starting point we used a double mutant of subtilisin BPN' (N62D/ G166D) that showed substantial preference for cleaving after sequences having consecutive dibasic residues (namely, at the P1 and P2 substrate positions) [Ballinger et al. (1995) Biochemistry 34, 13312-13319]. Additional specificity for basic residues was engineered at the P4 position by introducing subtilisin-to-furin substitutions at three hydrophobic residues that composed the S4 subsite (Y104, I107, and L126). Initial attempts to incorporate a Y104D or I107E mutation or the Y104D/I107E double mutation into the dibasic specific enzyme failed to generate the processed enzyme. The problem was traced to the inability of the mutant prosubtilisins to process themselves and fold correctly. Replacing the natural processing site sequence (AHAY) with a good furin substrate sequence (RHKR) resulted in expression of the triple subtilisin mutant (N62D/Y104D/G166D) we call "furilisin". Furilisin hydrolyzes synthetic tribasic substrates (succinyl-RAKR-pNA or succinyl-KAKR-pNA) with high catalytic efficiency (kcat/K(m) > 3 x 10(5) M-1 s-1) and discriminates in favor of Arg versus Ala at the P4 position by a factor of 360. The overall specificity change versus the wild-type enzyme was dramatic. For example, succinyl-RAKR-pNA was cleaved approximately 60000 times faster than succinyl-AAPF-pNA, a good substrate for wild-type subtilisin. Similarly, furilisin was inhibited (K1* = 29 nM) by a variant of the turkey ovomucoid third domain inhibitor that contained an engineered furin substrate site (RCKR decreases) [Lu et al. (1993) J. Biol. Chem. 268, 14583-14585] and not by one having a good wild-type subtilisin substrate sequence (ACTL decreases). Interestingly, the extreme changes in substrate specificity resulted from substantial synergy between the engineered subsites. These studies provide a basic example of how to manipulate substrate specificity in a modular fashion, thereby creating an engineered-enzyme that may be useful as a protein processing tool.

Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-beta by cathepsin D

Amyloid beta (A beta) is a 39-43-residue protein that originates from proteolysis of the beta-protein precursor (beta PP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant beta PP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant beta PP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH2-terminal sequence of A beta from wild-type beta PP, the double mutant, and the two single substitutions. Proteinase activity in brain extract cleaved the mutant substrate 100-fold faster than the wild-type substrate and the partial mutants 25-fold faster. The major cleavage site in all substrates was at the amyloidogenic Asp1 site. The brain activity appeared to be cathepsin D (CD), as indicated by similarities to purified CD in 1) the rate and site of substrates cleavage, 2) the pH optima, and 3) the sensitivity to pepstatin A. The increased activity against the mutant substrate was not shared by cathepsins B and C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furthermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr43 and Ala42, at ratios of 68% and 32%, respectively. CD degraded A beta 1-40 into six fragments but A beta 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavages resulting in A beta production and that it may prove to be a suitable therapeutic target.

Specificity of ribozyme designed for mutated DHFR mRNA

When MOLT-3 human acute leukemia cells were exposed sequentially to trimetrexate (TMQ) and then to methotrexate (MTX), the cells became resistant to antifolate. We designated this subline MOLT-3/TMQ 800-MTX 10,000. This cell line was found to contain two point mutations in the dihydrofolate reductase ( DHFR) gene: a T → C transition at nucleotide 95 in codon 31, and a T → A transition at nucleotide 100 in codon 33. In an attempt to specifically inhibit these double-mutated cells, we synthesized a ribozyme which perfectly base-paired with the double-mutated DHFR mRNA. We found that the ribozyme for the double-mutated DHFR mRNA not only cleaved the mutated DHFR RNA, but also efficiently cleaved the wild-type RNA substrate. This observation suggests proceeding with caution when using a ribozyme against a mutated mRNA of an essential enzyme as a specific means of treatment.

The Role of cosB, the Binding Site for Terminase, the DNA Packaging Enzyme of Bacteriophage λ in the Nicking Reaction

cosB is the binding site for terminase, the DNA packaging enzyme of phage λ, and cosN is the adjacent site at which terminase introduces staggered nicks to generate mature λ DNA molecules. There are three binding sites (R3, R2 and R1) within cosB for gpNu1, the small subunit of terminase. A particular transition mutation of R1, known to weaken binding of gpNu1 to R1, has been introduced into the other R sites, and in the present work the effects of R site mutations on nicking of cosN have been examined. Nicking experiments performed in the presence of ATP suggest that the most profound cosB mutation tested (the R3-R2-R1- mutation) would, at most, reduce cos nicking to ≃30% of the level observed for the wild-type substrate. In the presence of ATP, the R3-R2-R1- mutation had no significant effect on terminase nicking of the l strand and reduced r-strand nicking to 35% of the wild-type level. The other cosB mutations had no effect on the nicking of either DNA strand when nucleotide was added, but in the absence of ATP, most of the cos mutations resulted in some form of cosN nicking defect; the nicking defects, however, are milder than the in vivo packaging defects that result from the mutations. Quantitatively, only the effect of the R3-R2-R1- mutation on in vitro cosN nicking is reflective of the growth defect exhibited by a R3-R2-R1- phage but the nicking defect is only observed when ATP is omitted from the reaction. The proposal that the cosB mutations primarily affect DNA packaging rather than cosN nicking is discussed.All of the cosB mutations affect r-strand nicking to a greater extent than l-strand nicking, implying that the interaction of terminase with the left half of cosN occurs via the direct recognition of cosNL by terminase.The level of DNA substrate required for half-maximal cos nicking is approximately equivalent for reactions performed in the presence or absence of ATP, indicating that ATP does not increase the affinity of terminase for cosB . ATP does accelerate the rate of cos nicking, suggesting that the role of ATP in promoting nicking of the cosB- DNAs is primarily to increase the rate of conversion of a cosN-terminase complex into product. A possible fourth R site, R4, is located on the other side of cosN from cosB. The R4- mutation was found not to have any apparent effect on the ability of terminase to nick either DNA strand in the presence or absence of ATP.

Apolipoprotein B mRNA editing is associated with UV crosslinking of proteins to the editing site.

Apolipoprotein (apo) B100 mRNA undergoes editing of C-6666 to a U residue, which generates a stop-translation codon and defines the carboxyl terminus of apoB48. To aid purification of the editing enzyme we have undertaken UV crosslinking of a 32P-labeled substrate for apoB mRNA editing in vitro to proteins in an enterocyte editing extract. Proteins of 60 (p60) and 43 (p43) kDa, prominent among crosslinking bands, were competed for by unlabeled substrate, but not by nonspecific RNA, and did not crosslink to antisense RNA. Editing in vitro and UV crosslinking were inhibited by NaCl and vanadyl ribonucleoside complexes and by chemical modification of sulfhydryl, imidazolium, and guanidinium groups on the protein. The editing activity copurified predominantly with p60. To define the binding site for p60 on the substrate RNA, a series of scanning and point mutant RNAs, previously used to define nucleotides 6671-6681 as essential for editing, were used in competition studies with wild-type substrate. Results demonstrated that p60 binding is centered on nucleotides 6671-6674. We suggest that p60 contains the RNA-recognition component of the apoB mRNA-editing enzyme.

Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates.

Substrate recognition by the hairpin ribozyme has been proposed to involve two short intermolecular helices, termed helix 1 and helix 2. We have used a combination of three methods (cleavage of mismatched substrates, in vitro selection, and site-specific mutational analysis) to systematically determine the substrate recognition rules for this RNA enzyme. Assays measuring substrate cleavage in trans under multiple turnover conditions were conducted using the wild-type ribozyme and substrates containing mismatches in all sites potentially recognized by the ribozyme. Molecules containing single- and multiple-base mismatches in helix 2 at sites distant from the cleavage site (g-4c, u-5a, g-4c: u-5a) were cleaved with reduced efficiency, whereas those with mismatches proximal to the cleavage site (c-2a, a-3c, c-2a: a-3c) were not cut. Analogous results were obtained for helix 1, where mismatches distal from the cleavage site (u+7a, u+8a, u+9a, u+7a: u+8a: u+9a) were used much more efficiently than those proximal to the cleavage site (c+4a, u-5a, g+6c, c+4a: u+5a: g+6c). In vitro selection experiments were carried out to identify active variants from populations of molecules in which either helix 1 or helix 2 was randomized. Results constitute an artificial phylogenetic data base that proves base-pairing of nucleotides at five positions within helix 1 and three positions within helix 2 and reveals a significant sequence bias at 3 bp (c+4.G6, c-2.G11, and a-3.U12). This sequence bias was confirmed at two sites by measuring relative cleavage rates of all 16 possible dinucleotide combinations at base pairs c+4.G6 and c-2.G11.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa

PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD.

Complex Recognition Site for the Group I Intron-Encoded Endonuclease I-SceII

We have characterized features of the site recognized by a double-stranded DNA endonuclease, I-SceII, encoded by intron 4 alpha of the yeast mitochondrial COX1 gene. We determined the effects of 36 point mutations on the cleavage efficiency of natural and synthetic substrates containing the Saccharomyces capensis I-SceII site. Most mutations of the 18-bp I-SceII recognition site are tolerated by the enzyme, and those mutant sites are cleaved between 42 and 100% as well as the wild-type substrate is. Nine mutants blocked cleavage to less than or equal to 33% of the wild-type, whereas only three point mutations, G-4----C, G-12----T, and G-15----C, block cleavage completely. Competition experiments indicate that these three substrates are not cleaved, at least in part because of a marked reduction in the affinity of the enzyme for those mutant DNAs. About 90% of the DNAs derived from randomization of the nucleotide sequence of the 4-bp staggered I-SceII cleavage site are not cleaved by the enzyme. I-SceII cleaves cloned DNA derived from human chromosome 3 about once every 110 kbp. The I-SceII recognition sites in four randomly chosen human DNA clones have 56 to 78% identity with the 18-bp site in yeast mitochondrial DNA; they are cleaved at least 50% as well as the wild-type mitochondrial substrate despite the presence of some substitutions that individually compromise cleavage of the mitochondrial substrate. Analysis of these data suggests that the effect of a given base substitution in I-SceII cleavage may depend on the sequence at other positions.

Complex recognition site for the group I intron-encoded endonuclease I-SceII.

We have characterized features of the site recognized by a double-stranded DNA endonuclease, I-SceII, encoded by intron 4 alpha of the yeast mitochondrial COX1 gene. We determined the effects of 36 point mutations on the cleavage efficiency of natural and synthetic substrates containing the Saccharomyces capensis I-SceII site. Most mutations of the 18-bp I-SceII recognition site are tolerated by the enzyme, and those mutant sites are cleaved between 42 and 100% as well as the wild-type substrate is. Nine mutants blocked cleavage to less than or equal to 33% of the wild-type, whereas only three point mutations, G-4----C, G-12----T, and G-15----C, block cleavage completely. Competition experiments indicate that these three substrates are not cleaved, at least in part because of a marked reduction in the affinity of the enzyme for those mutant DNAs. About 90% of the DNAs derived from randomization of the nucleotide sequence of the 4-bp staggered I-SceII cleavage site are not cleaved by the enzyme. I-SceII cleaves cloned DNA derived from human chromosome 3 about once every 110 kbp. The I-SceII recognition sites in four randomly chosen human DNA clones have 56 to 78% identity with the 18-bp site in yeast mitochondrial DNA; they are cleaved at least 50% as well as the wild-type mitochondrial substrate despite the presence of some substitutions that individually compromise cleavage of the mitochondrial substrate. Analysis of these data suggests that the effect of a given base substitution in I-SceII cleavage may depend on the sequence at other positions.

Configuration of DNA strands and mechanism of strand exchange in the Hin invertasome as revealed by analysis of recombinant knots.

The Hin recombinase of Salmonella normally catalyzes a site-specific DNA inversion reaction that is very efficient when the Fis protein and a recombinational enhancer sequence are present. The mechanism of this recombination reaction has been investigated by analyzing the formation and structure of knots generated in different plasmid substrates in vitro. Hin seldom knots the wild-type substrate under standard recombination conditions. However, we show that increasing the length of DNA between the recombination sites and the enhancer and changing the sequence of the core nucleotides where strand exchange occurs increases the efficiency of the knotting reaction. The structure of the knots generated by different mutant substrates strongly supports a model involving a unique configuration of DNA strands at synapsis and DNA strand exchange mediated by rotation of one set of Hin subunits after DNA cleavage. Analysis of the stereostructure of the knots by electron microscopy of RecA-coated DNA molecules demonstrates that the direction of subunit rotation is exclusively clockwise. Because multiple subunit rotations generating knotted molecules do not occur efficiently when the enhancer is located in its native position, we suggest that the enhancer normally remains associated with the two recombination sites in the invertasome structure during strand exchange to limit strand rotation once it has been initiated. Under certain conditions, however, complex knots are formed that are probably the result of the premature release of the enhancer and multiple, unrestrained subunit exchanges.

The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA.

Commitment complexes contain U1 snRNP as well as pre-mRNA and are the earliest functional complexes that have been described during in vitro spliceosome assembly. We have used a gel retardation assay to analyze the role of the yeast pre-mRNA cis-acting sequences in commitment complex formation. The results suggest that only a proper 5' splice site sequence is required for efficient U1 snRNA-pre-mRNA complex formation. A role for the highly conserved UACUAAC branchpoint sequence is indicated, however, by competition experiments and by the direct analysis of branchpoint mutant substrates, which cannot form one of the two commitment complex species observed with wild-type substrates. The results suggest that the formation of a U1 snRNP-pre-mRNA complex is not dependent upon the presence of a branchpoint sequence but that the branchpoint sequence is recognized prior to U2 snRNP addition during in vitro spliceosome assembly.

Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition.

The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC. We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity. In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites). These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure. In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200). We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission.

Mutational analysis of a native substrate of the human immunodeficiency virus type 1 proteinase

Proteolytic processing of the gag/pol precursor by the human immunodeficiency virus type 1 proteinase is essential for the production of infectious viral particles. Although the sites of virus-specific cleavages have been determined, the primary amino acid sequences surrounding these sites are heterogeneous and the determinants that direct the cleavage specificity exhibited by human immunodeficiency virus type 1 proteinase remain largely undefined. We performed mutational analysis of the Tyr/Pro site, which produces the amino terminus of the viral capsid protein, and the Phe/Pro site, which produces the amino terminus of the proteinase. Mutations were made in a clone encoding a frameshift mutation that results in the expression of equimolar amounts of the substrate and proteinase in the form of a truncated gag/pol precursor. After single-amino-acid substitutions were made, their effects on proteolytic processing were examined by in vitro transcription and in vitro translation of the synthetic mRNA; translation products were then processed by exogenously added purified proteinase. Single-amino-acid substitutions yielded both substrates which were processed with wild-type efficiency and substrates on which processing was impaired. At the Tyr/Pro site in gag, processing was severely inhibited by substitutions within the P4, P2, P1, and P2' positions. The Phe/Pro site in pol, however, demonstrated far greater tolerance to amino acid substitution. These data suggest that the primary amino acid sequence around a scissile bond is more critical for cleavage of the Tyr/Pro site than the Phe/Pro site.

Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holo-ACP synthase.

Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.

Characterization of Holliday structures in FLP protein-promoted site-specific recombination.

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.

Characterization of Holliday Structures in FLP Protein-Promoted Site-Specific Recombination

Holliday structures are formed in the course of FLP protein-promoted site-specific recombination. Here, we demonstrate that Holliday structures are formed in reactions involving wild-type substrates and that they are kinetically competent with respect to the overall reaction rate. Together with a previous demonstration of chemical competence (L. Meyer-Leon, L.-C. Huang, S. W. Umlauf, M. M. Cox, and R. B. Inman, Mol. Cell. Biol. 8:3784-3796, 1988), Holliday structures therefore meet all criteria necessary to establish that they are obligate reaction intermediates in FLP-mediated site-specific recombination. In addition, kinetic evidence suggests that two distinct forms of the Holliday intermediate are present in the reaction pathway, interconverted in an isomerization process that is rate limiting at 0 degree C.

Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates

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 TyrSu3, tRNA TyrSu3A15, 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 TyrSu3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher K m compared to the precursor to tRNA TyrSu3. The precursor to tRNA TyrSu3A2, 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 TyrSu3 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 k cat compared to wild-type, which can explain its phenotype in vivo.