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At least 1,441 records · Page 80Linked to original sources
PubMed1984

Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region.

Transcription of the tryptophan (trp) operon of Escherichia coli and other bacterial species is regulated by the formation of alternative secondary structures in the leader segment of the transcript. During in vitro transcription of the trp leader region RNA polymerase pauses at base pair 92 after synthesis of an RNA hairpin secondary structure. We studied the dependence of pausing on hairpin stability by examining mutant trp templates containing base pair substitutions in the region corresponding to the hairpin secondary structure. Base changes that lower the stability of the hairpin were found to reduce both the frequency and half-life of RNA polymerase pausing while base changes that do not affect hairpin stability had little effect on pausing. Pausing was enhanced by the nusA protein; this enhancement was greatly reduced on mutant templates specifying less stable hairpins. The frequency of pausing on some mutant templates was correlated with the extent of read-through transcription beyond the trp attenuator, suggesting a possible role for pausing in the coupling of transcription and translation during transcription of the leader region of the operon.

DNA-Directed RNA Polymerases↗
PubMed2004

A mutation in polynucleotide phosphorylase from Escherichia coli impairing RNA binding and degradosome stability.

Polynucleotide phosphorylase (PNPase), a 3' to 5' exonuclease encoded by pnp, plays a key role in Escherichia coli RNA decay. The enzyme, made of three identical 711 amino acid subunits, may also be assembled in the RNA degradosome, a heteromultimeric complex involved in RNA degradation. PNPase autogenously regulates its expression by promoting the decay of pnp mRNA, supposedly by binding at the 5'-untranslated leader region of an RNase III-processed form of this transcript. The KH and S1 RNA-binding domains at the C-terminus of the protein (amino acids 552-711) are thought to be involved in pnp mRNA recognition. Here we show that a G454D substitution in E.coli PNPase impairs autogenous regulation whereas it does not affect the catalytic activities of the enzyme. Although the mutation maps outside of the KH and S1 RNA-binding domains, analysis of the mutant protein revealed a defective RNA binding, thus suggesting that other determinants may be involved in PNPase-RNA interactions. The mutation also caused a looser association with the degradosome and an abnormal electrophoretic mobility in native gels. The latter feature suggests an altered structural conformation of PNPase, which may account for the properties of the mutant protein.

Amino Acid Substitution↗
PubMed1997

Contribution of the C-terminal tail of U1A RBD1 to RNA recognition and protein stability.

The N-terminal RNA binding domain (RBD1) of the human U1A protein interacts specifically with a short RNA hairpin containing the U1 snRNA stem/loop II sequence. Previous RNA binding studies have suggested that the C-terminal tail of RBD1 contributes to RNA recognition in addition to interactions on the beta-sheet surface of the protein. To evaluate the contributions of these C-terminal residues in RBD1 to RNA binding affinity and specificity, as well as to study the thermodynamic stability of RBDs, a number of RBD1 mutants with truncated tails, with single amino acid substitutions, and with both a truncation and an amino acid substitution, have been constructed. The thermodynamic stabilities of these mutants have been measured and compared by GdnHCI unfolding experiments. The RNA binding affinity and specificity of these mutant proteins have been assessed by measuring the binding of each protein to the wild-type RNA hairpin and to selected RNA mutants with nucleotide substitutions in the RNA loop. The results demonstrate first that, although the C-terminal tail of RBD1 makes significant contributions to RNA binding affinity, it is not required for RNA binding, and second, its contributions to binding specificity are mediated only through selected nucleotides in the RNA loop, for in the absence of the tail, the protein continues to use other nucleotides to discriminate among RNAs. In these truncated proteins, the secondary structure intrinsic to the C-terminal tail is absent, yet their affinity and discrimination for RNAs are not lost. Thus, a structured tail is not required for RNA recognition.

Amino Acid Sequence↗
PubMed1995

Relative thermodynamic stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure.

Fourteen oligonucleotides 8-21 nucleotides in length and their complements were synthesized as DNA and RNA. For each sequence, four kinds of duplexes, DNA:DNA, RNA:RNA, DNA:RNA, and RNA:DNA, were prepared. Twelve sequences had A.T/U content varying from 25 to 80% and dPy content in the DNA strands varying from 0 to 100%. Thermodynamic stabilities of four duplexes for each sequence were determined in solution containing 100 mM Na+, 10 mM phosphate, and 0.1 mM EDTA, pH 7.1. CD spectra and electrophoretic mobility on native polyacrylamide gel were measured for most duplexes. Quantitative correlations of hybrid stability both with deoxypyrimidine content and, at fixed dPy content, with the fraction of A.T/U in duplexes were found. We also demonstrated that hybrids with 70-80% deoxypyrimidine DNA strand and a high or moderate A.T/U fraction displayed the highest relative stability compared to their RNA counterparts. Relationships of relative intensities of CD bands at 210 nm and relative electrophoretic mobilities of hybrids with relative hybrid stability suggested that hybrid conformation varies continuously between A- and B-form and is the decisive factor in relative hybrid stability.

Base Sequence↗
PubMed2001

Bacteriophage T7 protein kinase phosphorylates RNase E and stabilizes mRNAs synthesized by T7 RNA polymerase.

The T7 protein encoded by the early gene 0.7 exhibits bifunctional activity. Whereas its C-terminal one-third participates in host transcription shut-off, the N-terminal two-thirds bears a protein kinase ('PK') activity that can phosphorylate a number of host proteins in addition to itself. Here, we show that, when PK is expressed in uninfected Escherichia coli cells, the C-terminal half of RNase E and the associated RNA helicase RhlB are heavily phosphorylated. Meanwhile, a subset of RNase E substrates, including the lac and cat mRNAs synthesized by bacteriophage T7 RNA polymerase (RNAP), are stabilized. These mRNAs are genuinely less stable than their counterparts synthesized by E. coli RNAP, because T7 RNAP outpaces translating ribosomes, creating naked, RNase E-sensitive mRNA stretches behind itself. Thus, PK alleviates this effect of desynchronizing transcription and translation. The relationship between the modification of RNase E and RhlB and these mRNA stabilization effects, which may be relevant to the stability of late T7 mRNAs during infection, is discussed.

DNA-Directed RNA Polymerases↗
PubMed2000

H19 gene expression is up-regulated exclusively by stabilization of the RNA during muscle cell differentiation.

H19 is a paternally imprinted gene whose expression produces a 2.4 kb RNA in most tissues during development and in mammalian myoblastic cell lines upon differentiation. Deletion of the active maternal allele of H19 and its flanking regions in the mouse leads to biallelic methylation and loss of imprinting of the neighbouring Igf2 gene. The function of H19 RNA remains unknown and, although polysome-associated, the absence of a conserved open reading frame suggests that it does not encode a protein product. We describe a novel post-transcriptional regulation of H19 gene expression which, in spite of this lack of coding capacity, is dependent on translational activity. We show that stabilization of the RNA is solely responsible for its accumulation during in vitro muscle cell differentiation. This conclusion is based on the finding that inhibition of protein synthesis results in a dramatic destabilization of H19 RNA in proliferating mouse C2C12 myoblastic cells but not in differentiated cells, and on run-on experiments which showed that the rate of transcription of H19 RNA remains constant during muscle cell differentiation. This mechanism could also be involved in H19 gene expression during mouse development in addition to its transcriptional activation which we have shown to occur.

Animals↗
PubMed2003

The apical loop of the HIV-1 TAR RNA hairpin is stabilized by a cross-loop base pair.

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive. We performed biochemical studies and molecular dynamics simulations, which indicate that the TAR loop is structured and stabilized by a cross-loop base pair between residues C30 and G34. Mutational disruption of the cross-loop base pair results in reduced Tat response of the LTR promoter, which can be rescued by compensatory mutations that restore the base pair. Thus, Tat-mediated transcriptional activation depends on the structure of the TAR apical loop. The C30-G34 cross-loop base pair classes TAR in a growing family of hairpins with a structured loop that was recently identified in ribosomal RNA, tRNA, and several viral and cellular mRNAs.

2-Aminopurine↗
PubMed1993

Effects of amino acid substitution on the thermal stability of MS2 capsids lacking genomic RNA.

The thermal stability of capsids of the bacteriophage MS2, lacking genomic RNA, has been investigated using electron microscopy. Coat protein mutants with amino acid substitutions at residues involved in making contracts at both inter-molecular interfaces and within the coat protein submit are also capable of forming 'empty' capsids of the same size and symmetry as the wild-type protein. Mutations have been characterised which are neutral, deleterious or advantageous in terms of thermal stability. In some cases, the results can be rationalised by reference to the recently refined X-ray crystal structure of the wild-type particle.

Amino Acids↗
PubMed2004

Sequential dimerization of human zipcode-binding protein IMP1 on RNA: a cooperative mechanism providing RNP stability.

Active cytoplasmic RNA localization depends on the attachment of RNA-binding proteins that dictate the destination of the RNA molecule. In this study, we used an electrophoretic mobility-shift assay in combination with equilibrium and kinetic analyses to characterize the assembly of the human zipcode-binding protein IMP1 on targets in the 3'-UTR from Igf-II mRNA and in H19 RNA. In both cases, two molecules of IMP1 bound to RNA by a sequential, cooperative mechanism, characterized by an initial fast step, followed by a slow second step. The first step created an obligatory assembly intermediate of low stability, whereas the second step was the discriminatory event that converted a putative RNA target into a 'locked' stable RNP. The ability to dimerize was also observed between members of the IMP family of zipcode-binding proteins, providing a multitude of further interaction possibilities within RNP granules and with the localization apparatus.

3' Untranslated Regions↗
PubMed2002

The RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter.

The RNA polymerase of the influenza virus is responsible for the transcription and replication of the segmented RNA viral genome during infection of host cells. Polymerase function is known to be strictly dependent on interaction with its RNA promoter, but no attempts to investigate whether the virion RNA (vRNA) promoter stabilizes the polymerase have been reported previously. Here we tested whether the vRNA promoter protects the polymerase against heat inactivation. We prepared partially purified recombinant influenza A virus RNA polymerase, in the absence of influenza virus vRNA promoter sequences, by transient transfection of expression plasmids into human kidney 293T cells. The polymerase was found to be heat labile at 40 degrees C in the absence of added vRNA. However, it was protected from heat inactivation if both the 5' and 3' strands of the vRNA promoter were present. By using the ability of vRNA to protect the enzyme against heat inactivation, we established a novel assay, in conjunction with a mutagenic approach, that was used to test the secondary structure requirement of the vRNA promoter for polymerase binding. Binding required a panhandle structure and the presence of local hairpin loop structures in both the 5' and 3' ends of vRNA, as suggested by the corkscrew model. The interaction of the vRNA promoter with the influenza virus RNA polymerase heterotrimeric complex is likely to favor a particular closed conformation of the complex, thereby ensuring the stability of the RNA polymerase within both the infected cell and the isolated virus.

Base Pairing↗
PubMed1978

Stability of polyadenylated RNA in differentiating myogenic cells.

Three independent methods of measurement showed that cytoplasmic polyadenylated RNA from the differentiating myogenic cell line L8 consists of two main populations with regard to stability, one with a half-life of less than 4 h and the other with a half-life of 17--54 h. Similar results were obtained in the presence and absence of actinomycin D. During the fusion of mononucleated myoblasts into multinucleated fibers, there was an increase in both the steady-state pool of the more stable polyadenylated RNA and the proportion of stable polyadenylated RNA synthesized in pulse labelling.

Animals↗
PubMed1998

Hypoxic regulation of VEGF mRNA stability by RNA-binding proteins.

Vascular endothelial growth factor (VEGF), is a potent angiogenic factor whose expression is dramatically induced by hypoxia. We have previously demonstrated that the induction of VEGF by hypoxia is in large part the result of an increased stability of VEGF mRNA. The stabilization of VEGF mRNA by hypoxia is mediated by the binding of sequence-specific RNA-binding proteins. This review focuses on one such protein, HuR, an RNA-binding protein which we have recently shown is critical for the hypoxic stabilization of VEGF mRNA.

Journal Article↗
PubMed2005

Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco.

Matrix attachment regions (MARs) are DNA elements that can increase and stabilize transgene expression. We investigated the effect of the RB7 MAR on transgenic virus resistance. Constructs for resistance to tomato spotted wilt virus (TSWV) with and without flanking RB7 MARs were used to transform tobacco and produce homozygous lines. The population with the MAR construct had a significantly higher percentage of TSWV resistant plants in the R1 generation than the nonMAR population. Each resistant line was advanced to the R4 generation, and significantly fewer MAR lines lost resistance over generations compared to the nonMAR population. Lines with TSWV resistance in growth chamber tests were also resistant in field trials. Two lines that were resistant in the R1 generation and susceptible in the R4 were examined in more detail in order to determine if transcriptional silencing of the transgene was occurring in the later generation. Short interfering 21-25 nt RNAs from the transgene that are characteristic of post-transcriptional gene silencing (PTGS) were present in the resistant R1 plants, but not the susceptible R4 plants, indicating that virus resistance was associated with PTGS of the transgene. Loss of resistance was accompanied by an increase in promoter methylation in both lines. In line M41, the transgene was fully silenced at the transcriptional level in the R4 as shown by nuclear run-on assays. In line NM13, transgene transcription and RNA accumulation was still present in the R4 generation, but the level of transcription was not sufficient to trigger PTGS, suggesting that this line may have partial transcriptional silencing. These results are consistent with the concept that MARs may prevent transcriptional silencing.

Breeding↗
PubMed2005

A single intermolecular contact mediates intramolecular stabilization of both RNA and protein.

An arginine-rich peptide from the Jembrana disease virus (JDV) Tat protein is a structural "chameleon" that binds bovine immunodeficiency virus (BIV) or HIV TAR RNAs in two different binding modes, with an affinity for BIV TAR even higher than the cognate BIV peptide. We determined the NMR structure of the JDV Tat-BIV TAR high-affinity complex and found that the C-terminal tyrosine in JDV Tat forms a network of inter- and intramolecular hydrogen bonding and stacking interactions that simultaneously stabilize the beta-hairpin conformation of the peptide and a base triple in the RNA. A neighboring histidine also appears to help stabilize the peptide conformation. Induced fit binding is recurrent in protein-protein and protein-nucleic acid interactions, and the JDV Tat complex demonstrates how high affinity can be achieved not only by optimization of the binding interface but also by inducing new intramolecular contacts that stabilize each binding partner. Comparison to the cognate BIV Tat peptide-TAR complex shows how such a costabilization mechanism can evolve with only small changes to the peptide sequence. In addition, the bound structure of BIV TAR in the chameleon peptide complex is strikingly similar to the bound conformation of HIV TAR, suggesting new strategies for the development of HIV TAR binding molecules.

Arginine↗
PubMed1984

Basepairing of oligonucleotides to the 3' end of 16S ribosomal RNA is not stabilized by ribosomal proteins.

We have studied the binding of the octanucleotide (5'-3')d(AAGGAGGT) which is fully complementary to the 3' end of 16S ribosomal RNA, to ribosomes and to the isolated target sequence (5'-3') (ACCUCCUUA). The binding constant for 30S or 70S ribosomes is (5 +/- 2) X 10(7) mol-1, whereas the duplex containing the octa- and the nonanucleotide has an association constant of (6 +/- 3) X 10(7) mol-1. The two values are the same within the experimental error. This result suggests that basepairing at the 3' end of 16S rRNA is not stabilized by ribosomal proteins.

Base Composition↗