Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea.

The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES7) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES7 and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES7) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES7 and CMC-GroES7 are decreased with a half-time of (23 ±â€¯2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ~ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.

Removal of protein S1 from Escherichia coli ribosomes without the use of affinity chromatography.

The paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion. To avoid co-sedimentation of the S1-bound polypyrimidine with the ribosomes, its length should not exceed several hundred nucleotides. Unlike popular affinity chromatography through a poly(U) Sepharose or poly(U) cellulose column, the method tolerates limited polyribonucleotide degradation by eventual traces of ribonucleases, and can readily be incorporated into standard protocols for the isolation of ribosomes by centrifugation.

Ribosomal protein S1 functions as a termination factor in RNA synthesis by Qß phage replicase.

S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qß replicase, the RNA-directed RNA polymerase of bacteriophage Qß. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qß replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.

Functional circularity of legitimate Qbeta replicase templates.

Qbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs in vitro. Previous studies have shown that Qbeta replicase can initiate and elongate on a variety of RNAs; however, only a minute fraction of them are recognized as 'legitimate' templates. Guanosine 5'-triphosphate (GTP)-dependent initiation on a legitimate template generates a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid, a powerful inhibitor of RNA-protein interactions. On the contrary, initiation on an illegitimate template is GTP independent and does not result in the aurintricarboxylic-acid-resistant replicative complex. This article demonstrates that the 3' and 5' termini of a legitimate template cooperate during and after the initiation step. Breach of the cooperation by dividing the template into fragments or by introducing point mutations at the 5' terminus reduces the rate and the yield of initiation, increases the GTP requirement, decreases the overall rate of template copying, and destabilizes the postinitiation replicative complex. These results revive the old idea of a functional circularity of legitimate Qbeta replicase templates and complement the increasing body of evidence that functional circularity may be a common property of RNA templates directing the synthesis of either RNA or protein molecules.

Factors influencing RNA degradation by Thermus thermophilus polynucleotide phosphorylase.

At the optimal temperature (65 degrees C), Thermus thermophilus polynucleotide phosphorylase (Tth PNPase), produced in Escherichia coli cells and isolated to functional homogeneity, completely destroys RNAs that possess even a very stable intramolecular secondary structure, but leaves intact RNAs whose 3' end is protected by chemical modification or by hybridization with a complementary oligonucleotide. This allows individual RNAs to be isolated from heterogeneous populations by degrading unprotected species. If oligonucleotide is hybridized to an internal RNA segment, the Tth PNPase stalls eight nucleotides downstream of that segment. This allows any arbitrary 5'-terminal fragment of RNA to be prepared with a precision similar to that of run-off transcription, but without the need for a restriction site. In contrast to the high Mg(2+) requirements of mesophilic PNPases, Tth PNPase retains significant activity when the free Mg(2+) concentration is in the micromolar range. This allows minimization of the Mg(2+)-catalysed nonenzymatic hydrolysis of RNA when phosphorolysis is performed at a high temperature. This capability of Tth PNPase for fully controlled RNA phosphorolysis could be utilized in a variety of research and practical applications.

Viral RNA-directed RNA polymerases use diverse mechanisms to promote recombination between RNA molecules.

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qbeta phage-derived RNA, such that if aligned at complementary 3'-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qbeta replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qbeta replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.

Qbeta replicase discriminates between legitimate and illegitimate templates by having different mechanisms of initiation.

Qbeta replicase (RNA-directed RNA polymerase of bacteriophage Qbeta) exponentially amplifies certain RNAs (RQ RNAs) in vitro. Here we characterize template properties of the 5' and 3' fragments obtained by cleaving one of such RNAs at an internal site. We unexpectedly found that, besides the 3' fragment, Qbeta replicase can copy the 5' fragment and a number of its variants, although they lack the initiator region of RQ RNA. This copying can occur as a 3'-terminal elongation or through de novo initiation. In contradistinction to RQ RNA and its 3' fragment, initiation on these templates occurs without regard to the 3'-terminal or internal oligo(C) clusters, is GTP-independent, and does not result in a stable replicative complex capable of elongation in the presence of aurintricarboxylic acid. The results suggest that, although Qbeta replicase can initiate and elongate on a variety of RNAs, only some of them are recognized as legitimate templates. GTP-dependent initiation on a legitimate template drives the enzyme to a "closed" conformation that may be important for keeping the template and the complementary nascent strand unannealed, without which the exponential replication is impossible. Triggering the GTP-dependent conformational transition at the initiation step could serve as a discriminative feature of legitimate templates providing for the high template specificity of Qbeta replicase.

Molecular colony diagnostics: detection and quantitation of viral nucleic acids by in-gel PCR.

When PCR is carried out in a polyacrylamide gel, each target molecule forms a molecular colony that comprises many copies of the original template. By counting the number of colonies, one can directly determine the target titer, with 100% of the DNA molecules and approximately 15% of the RNA molecules being detected. Furthermore, because of the spatial separation of the products in the gel, no interference is observedfrom another simultaneously amplified target even if it is present at a 106 higher amount orfrom human nucleic acids that outweigh the target by up to a factor of 1,012, which is often true of clinical samples. All these features provide for an accurate and reliable assay of viruses even at very low amounts, that is, in cases most important to diagnostics.