The structure of substrate-free microbial ribonuclease binase and of its complexes with 3'GMP and sulfate ions.

The structures of Bacillus intermedius ribonuclease (binase), an extracellular 109-residue enzyme, and its complexes with 3'GMP and sulfate ions were solved at 1.65 and 2.0 A, respectively. The structures were refined using REFMAC. The crystal of free binase belongs to the space group C2, whereas the crystals of complexes belong to the space group P2(1)2(1)2(1). In both crystal lattices the asymmetric unit contains two molecules which form an identical dimer. The structure of the dimer is such that only one of its subunits can bind the nucleotide in the 3'GMP-binase complex, where the guanyl base is located in the recognition loop of the enzyme. In both binase complex structures the phosphate group of 3'GMP or one of the sulfate ions make an electrostatic interaction with the binase molecule at the catalytic site. A second phosphate-binding site was found in the structures of the complexes at the cleft formed by the loop 34-39, the main chain of Arg82 and the side chain of Trp34. Comparison of the complex and unliganded enzyme crystal structures shows that there are some small but distinct differences in the specificity loop (56-62) and in the loops 34-39 and 99-104 associated with the binding of the nucleotide and sulfate ions.

A step subsequent to preinitiation complex assembly at the ribosomal RNA gene promoter is rate limiting for human RNA polymerase I-dependent transcription.

The assembly, disassembly, and functional properties of transcription preinitiation complexes (PICs) of human RNA polymerase I (Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that these complexes contained the factors required to support initiation of transcription, SL1, upstream binding factor (UBF), and Pol I. We have demonstrated that, throughout a single round of transcription, SL1 and UBF remain promoter bound. Moreover, the promoter-bound SL1 and UBF retain the ability to function in transcription initiation. SL1 has a central role in the stable association of the PIC with the promoter DNA. The polymerase component of the PIC is released from the promoter during transcription yet is efficiently recycled and able to reinitiate from "poised" promoters carrying SL1 and UBF, since the PICs captured on the immobilized templates sustained multiple rounds of transcription. Kinetic analyses of initiation of transcription by Pol I revealed that Pol I-dependent transcription is rate limited in a step subsequent to recruitment and assembly of Pol I PICs. The rate of RNA synthesis is primarily determined by the rates at which the polymerase initiates transcription and escapes the promoter, referred to as promoter clearance. This rate-limiting step in Pol I transcription is likely to be a major target in the regulation of rRNA gene expression.

hRRN3 is essential in the SL1-mediated recruitment of RNA Polymerase I to rRNA gene promoters.

A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry.

Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.

RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations.

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate. The extracellular ribonuclease from Bacillus intermedius (binase, RNase Bi) shares a common mechanism for RNA hydrolysis with mammalian RNases. Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage. Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GpC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied. The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.

Ribonuclease A mutant His119 Asn: the role of histidine in catalysis.

Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His12, His119, and Lys41) are known to be involved in catalysis. Mutation of His119 to asparagine was generated to study the role of His119 in RNase A catalysis. The mutant enzyme has been isolated and characterized. The mutation significantly decreases the rate of the transesterification reaction and has no effect on substrate affinity of the enzyme. An analysis of the enzymatic properties of H119N RNase A suggests that the imidazole ring of His119 of the wild-type enzyme must be protonated in an enzyme-substrate productive complex. Thus our results indicate that a contribution of protonated His119 into the catalysis is not restricted to protonation of oxygen atom of the substrate leaving group and that His119 participates directly in a transition state stabilization via hydrogen bonding.

Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase).

Members of the microbial guanyl-specific ribonuclease family show a high level of structural homology. The structural basis for guanyl base binding by microbial ribonucleases has been established for all members of the family and the existence of a guanine recognition loop was shown. However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides composed of more than 4 or 5 nucleotides. Using site-directed mutagenesis we introduced a number of amino acid substitutions into the base recognition loop of binase. The donor sequence originated from the guanyl specific ribonuclease Sa. Two single, two double and one triple (entire loop substitution) mutants were constructed and overproduced in E. coli. The kinetic properties of the mutant variants are different from the wild-type protein. Amino acid substitutions R61V, G60S, S56Q/R61V, G60S/R61V show 3-fold, 7-fold, 4-fold and 12-fold increased guanyl specificity respectively. However, all mutants retain the ability to catalyse the hydrolysis of a poly(A) substrate.

An efficient system for active bovine pancreatic ribonuclease expression in Escherichia coli.

Bovine pancreatic ribonuclease (RNase A) is a member of a homologous group of extensively studied proteins. It is a small, basic protein, containing 124 amino acid residues and four stabilizing disulfide bridges. Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids. Since this degradation of RNA interferes with normal cell functions, the signal peptide of alkaline phosphatase (phoA, Escherichia coli) was cloned onto the gene coding for RNase A, directing the protein to the periplasm. Several expression systems have been evaluated which use T7, trc, or PR promoters to transcribe the RNase A gene. Also, variation in host strains was tested to optimize the protein yield. It was found that the PR system gave better expression than the two other systems. E. coli strain BL21 was shown to be the strain in which export to the periplasm was most effective and recombinant RNase A could be isolated from the periplasmic fraction of these cells. The system provides a stable yield of active recombinant bovine pancreatic RNase of about 45-50 mg/liter of cell culture.

An improved system for ribonuclease Ba expression.

The extracellular ribonuclease from Bacillus amyloliquefaciens (barnase, RNase Ba) is a well-characterized enzyme extensively used in structure-function studies. A new system for efficient expression and purification of barnase has been developed. The strong regulated expression cassette with the Pr promoter of lambda phage and the cooperative expression of barnase and barstar under its control have been applied to expression of these proteins in Escherichia coli. The expression cassette containing the Pr promoter of E. coli lambda phage under cI repressor regulation and the nucleotide sequence coding for barnase and barstar structural genes were merged into the plasmid pTN441, which was used for large-scale barnase production. The phoA signal peptide was used to express the target protein into cell periplasm. The purification of RNase Ba was carried out in two steps: the initial sample was concentrated followed by RP-HPLC. The system provides a stable yield of homogeneous protein of about 100-150 mg per liter of culture medium.

[Barnase mutant Ser57Ala: preparation and properties]. / Mutant barnazy Ser57Ala: poluchenie i svoistva.

Barnase, an extracellular ribonuclease produced by Bacillus amyloliquefaciens, belongs to a family of small microbial ribonucleases with similar structure and properties. These enzymes hydrolyze phosphodiester bonds on the 3' side of guanosine nucleotides in RNA. The guanylic specificity of barnase is more pronounced in the hydrolysis of dinucleotides or cyclonucleotide phosphates as substrates than in the hydrolysis of RNA or polynucleotides. To have an insight into the molecular basis of this phenomenon, we mutated amino acid residue Ser-57 in the "base recognition loop" of RNase Ba. The mutant protein was expressed in Escherichia coli producing system and purified for the study of the kinetic properties in the cleavage polynucleotide reactions. It was shown that the mutation of amino acid residue Ser-57 for Ala in the "recognition loop" of RNase Ba does not significantly influence the kinetic parametres of hydrolysis of polynucleotide substrates.

[Superproduction of Bacillus intermedius 7P ribonuclease (binase) in Escherichia coli]. / Superproduktsiia ribonukleazy Bacillus intermedius 7P (binzay) v Escherichia coli.

We have reported previously about the cloning of the binase gene in E. coli. In this work, using an original approach named "homolog gene recombination" method (HGR), vectors for binase expression in E. coli have been constructed. Transcription of the binase gene have been directed through either tac-promoter or PR-promoter of bacteriophage lambda under the control of temperature-sensitive CI857 repressor. The last promoter gave the maximum yield of binase, up to 100 mg of protein per litre of heat-induced bacterial culture. The location of the transcription terminator at the 3' terminus of the binase gene raised the expression approximately two times more. A chromatographic method have been developed and applied for the control of binase accumulation in growth medium without measuring the ribonuclease activity.