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At least 307 records · Page 17Linked to original sources
PubMed1991

Accurate and efficient RNA polymerase II transcription with a soluble nuclear fraction derived from Drosophila embryos.

We describe the preparation and biochemical properties of a soluble nuclear fraction derived from Drosophila embryos. This extract, which can be easily prepared in 2.5 hr, is capable of accurate and efficient RNA polymerase II transcription of a variety of diverse genes from both Drosophila and mammals. With the relatively strong promoter of the Drosophila Krüppel gene, it is possible to achieve 20% template usage in a single round of transcription, which is considerably higher than the template usage of approximately 3% seen with standard nuclear extracts. Further, although U small nuclear RNA genes are refractory to transcription with HeLa transcription extracts, the soluble nuclear fraction transcribes a U1 small nuclear RNA gene from Drosophila. Moreover, transcriptional activation by sequence-specific activators can be attained in vitro with the soluble nuclear fraction. The overall transcriptional efficiency appears limited to 0.45 transcript per template of DNA per 30 min, but the mechanism of limitation is not known. The soluble nuclear fraction, which was developed to recreate the environment within the nucleus, should be useful when high efficiencies of RNA polymerase II transcription are desired.

Animals↗
PubMed2001

Structural properties of carnation mottle virus p7 movement protein and its RNA-binding domain.

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an alpha/beta RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7(17-35)), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent alpha-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNA-binding proteins and peptides. The low conserved N terminus of p7 (peptide p7(1-16)) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7(40-61)) adopts a beta-sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNA-binding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.

Amino Acid Sequence↗
PubMed2004

A simple method for improving protein solubility and long-term stability.

Increasing a protein concentration in solution to the required level, without causing aggregation and precipitation is often a challenging but important task, especially in the field of structural biology; as little as 20% of nonmembrane proteins have been found to be suitable candidates for structural studies predominantly due to poor protein solubility. We demonstrate here that simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (up to 8.7 times). These amino acids are effective in preventing protein aggregation and precipitation, and they dramatically increase the long-term stability of the sample; additionally, they protect protein samples from proteolytic degradation. Specific protein-protein and protein-RNA interactions are not adversely affected by the presence of these amino acids. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR.

Animals↗
PubMed2000

RseB binding to the periplasmic domain of RseA modulates the RseA:sigmaE interaction in the cytoplasm and the availability of sigmaE.RNA polymerase.

The Escherichia coli sigmaE regulon has evolved to sense the presence of misfolded proteins in the bacterial envelope. Expression of periplasmic chaperones and folding catalysts is under the control of sigmaE RNA polymerase. The N-terminal domain of RseA sequesters sigmaE in the cytoplasmic membrane, preventing its association with core RNA polymerase. The C-terminal domain of RseA interacts with RseB, a periplasmic protein. The relative concentration of sigmaE:RseA:RseB is 2:5:1 and this ratio remains unaltered upon heat shock induction of the sigmaE regulon. Purification from crude cellular extracts yields cytoplasmic, soluble sigmaE RNA polymerase as well as membrane sequestered sigmaE.RseA and sigmaE.RseA.RseB. RseB binding to the C-terminal domain of RseA increases the affinity of RseA for sigmaE by 2- to 3-fold (Kd 50-100 nM). RseB binds also to the misfolded aggregates of MalE31, a variant of maltose binding protein that forms inclusion bodies in the periplasm. We discuss a model whereby the RseB-RiseA interaction represents a measure for misfolded polypeptides in the bacterial envelope, modulating the assembly of sigmaE RNA polymerase and the cellular heat shock response.

ATP-Binding Cassette Transporters↗
PubMed1985

Bacterial lipopolysaccharide induction of ornithine decarboxylase in the macrophage-like cell line RAW264: requirement of an inducible soluble factor.

Ornithine decarboxylase (ODC, EC 4.1.1.17) activity is induced in the RAW264 macrophage-like cell line by bacterial lipopolysaccharide (LPS). As little as 0.1 ng/ml LPS promoted an increase in ODC activity, while maximal ODC activity (30-fold above control) was induced with 1.0 microgram/ml LPS. An increase in ODC activity was detectable within 90 min of LPS addition. The LPS-induced increase in ODC activity was prevented by inhibitors of protein and RNA synthesis. The induction of the enzyme by LPS was not dependent on prostaglandin production. However, PGE2 (1 microgram/ml) and 8-bromo-cyclic AMP (1 mM), neither of which had an effect on ODC activity when added alone, each acted synergistically to enhance the LPS induction of ODC activity. Enzyme induction was not associated with an alteration in Km for ornithine, which remained constant at 0.04 mM. The extent of the increase in ODC in response to LPS increased with increasing cellular density. This relationship was dependent not on absolute cell density of the monolayer but on the cell number in relation to medium volume, and this dependence could be extrapolated to the origin. Addition of conditioned media from LPS-stimulated but not unstimulated cultures enhanced the ODC increase in sparsely plated cultures in response to a maximal concentration of LPS. The addition of polymyxin B, a reagent that blocks the effects of LPS, including the increase in ODC activity, did not totally inhibit the conditioned medium stimulation. This data indicates that two signals, LPS and a LPS-induced mediator, are involved in the induction of ODC activity in RAW264 cells.

Animals↗
PubMed1966

Selection of sucrose-dependent Escherichia coli to obtain envelope mutants and fragile cultures.

Mutants isolated as sucrose-dependent include many with apparent envelope defects, which fact often leads to filamentous growth or lysis in the absence of sucrose. One mutant can grow exponentially in 4 percent sucrose, but is very fragile: it releases all of its RNA and soluble protein when treated with 0.5 percent sodium deoxycholate. These characteristics permit the study of unstable structures and rapid processes in actively growing cells.

Bile Acids and Salts↗
PubMed1978

Proteins bound to heterogeneous nuclear RNA of simian-virus-40-infected cells.

Heterogeneous nuclear RNA . protein (hnRNA . protein) complexes from simian-virus-40 (SV40)-infected cells late in infection contain 7--10% RNA sequences specific to SV40 DNA. The SV40 nuclear RNA . protein complexes sediment at 60--70 S. The reality and specificity of the RNA-protein association is shown in metrizamide gradients. Protein and RNA lebels of hnRNA . protein-particles in SV40-infected cells follow a parallel pattern with a peak at 1.28 g/cm2 whereas a mixture of ribosomal RNA and soluble cytoplasmic proteins is separated according to the different densities in metrizamide. Analysis of hnRNA . protein from infected cells by two-dimensional gel electrophoresis shows the presence of a number of new proteins. It is demonstrated that three of these proteins are cellular ones induced by the virus infection and hence constitute good candidates to be specific RNA . protein particles for virus nuclear RNA. The presence of actin in hnRNA . protein particles from normal and SV40-infected cells and the presence of the major capsid protein VP1 in hnRNA . protein particles from SV40-infected cells is discussed.

Base Sequence↗
PubMed1967

Structure and function of bacteriophage R17 replicative intermediate ribonucleic acid. II. Properties of the parental labeled molecule.

Replicative intermediate ribonucleic acid (RNA), designated RI, which contained parental RNA labeled with (32)P was separated by filtration through agarose from the nucleic acids prepared from (32)P-labeled RNA phage-infected Escherichia coli. A larger amount of ribonuclease-sensitive parental label was found in the rapidly sedimenting forms of RI than in the slower sedimenting forms, indicating that parental RNA is displaced to form a single-stranded tail. This result indicates that some phage RNA is generated by asymmetric semiconservative replication of RI, but it does not mean that a portion of the RI duplexes cannot be conserved during generation of phage RNA. Parental RNA was also found in double-stranded RNA with no apparent tails which sedimented with an S value of 13. This RNA was soluble in 2 m NaCl, and its sedimentation rate was unaffected by ribonuclease; nevertheless, single-strand scissions were produced by ribonuclease and were detected after the duplex was converted to its component single strands.

Coliphages↗