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At least 1,639 records · Page 91Linked to original sources
PubMed1985

Three-dimensional arrangement of the Escherichia coli 16 S ribosomal RNA.

A model for the arrangement of the Escherichia coli 16 S ribosomal RNA in the 30 S ribosomal subunit is given. This model is based on the 16 S ribosomal RNA secondary structure, intramolecular RNA crosslinking results, protein-RNA interactions, and the locations of proteins within the 30 S subunit. These considerations allow placement of most of the RNA helices in approximate positions. The overall shape (that of an asymmetric Y) is very reminiscent of the description of the shape of the RNA made by direct determinations and is reasonably correlated to the appearance of the 30 S subunit. The identities of the three major secondary-structure domains of the 16 S ribosomal RNA are, for the most part, preserved. In addition, many close contacts between the 5' and middle RNA domains occur in the body of the particle. The 3'-terminal domain is situated in the central part of the model. This position corresponds to the region between the head and the platform structure in the 30 S subunit. The regions that represent the general locations of the messenger RNA and transfer RNA binding sites can be identified in the model.

Cross-Linking Reagents↗
PubMed2002

Secondary structure and hybridization accessibility of hepatitis C virus 3'-terminal sequences.

The 3'-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3' untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3' terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3'-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3'-X region RNA in vitro.

5' Untranslated Regions↗
PubMed1996

Sequence variation as a strategy for crystallizing RNA motifs.

The determination of RNA structures by X-ray crystallography is an exciting and developing field. At present, the crystallographic characterization of RNA is limited by the difficulty in obtaining large, high quality crystals. This paper outlines several techniques for improving the likelihood of obtaining RNA crystals, for improving the size of those crystals, and for extending the limit of the diffraction maxima. Sequence variations have proven to be more effective in changing the quality of the crystals than variations in crystallization conditions, often making the difference between obtaining true single crystals and multiply twinned crystalline material.

Base Sequence↗
PubMed1997

Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3' end spacing distinguishes pause and termination pathways.

Transcription is delayed in the leader regions of the Escherichia coli trp and his operons by multipartite pause signals that consist of four components: a nascent RNA structure (the pause hairpin), the 10 or 11 nt 3'-proximal region between the pause hairpin and the RNA 3' end, the bases in the active site, and approximately 14 bp of duplex DNA downstream from the pause site. Results described in the accompanying paper suggest that the his pause hairpin slows nucleotide addition via interaction with an easily disordered surface on RNA polymerase. Here we report that the four pause signal components slow nucleotide addition in a single kinetic intermediate. Formation of the paused transcription complex, in contrast, involves synergistic effects of RNA and DNA sequences that select the wild-type pause site from among several adjacent possibilities. Extending the pause hairpin with one G x C base-pair reduces pausing, apparently by interfering with pause hairpin interaction; adding a second C x G base-pair that reduces the 3'-proximal RNA to 9 nt or less (within the 7 to 9 nt characteristic of rho-independent terminators) induces transcript release. We propose that escape from the pause is governed by a rate-limiting isomerization that may require substrate NTP binding to re-establish the active site geometry, whereas transcript release and termination ensue when the hairpin interaction is weakened and isomerization to an active conformation is blocked.

Bacterial Proteins↗
PubMed1999

Nucleotide analog interference mapping.

Single-atom substitution experiments provide atomic resolution biochemical information concerning RNA structure and function. Traditionally, these experiments are performed using chimeric RNAs generated by reassembly of full-length RNA from a synthetic substituted oligonucleotide and a truncated RNA transcript. Unfortunately, this technique is limited by the technical difficulty of assembling and measuring the effect of each singly substituted molecule in a given RNA. Here we review an alternate method for rapidly screening the effect of chemical group substitutions on RNA function. Nucleotide analog interference mapping is a chemogenetic approach that utilizes a series 5'-O-(1-thio)-nucleoside analog triphosphates to simultaneously, yet individually, probe the contribution of a functional group at every nucleotide position in an RNA molecule. A population of randomly substituted RNAs is prepared by including phosphorothioate-tagged nucleotide analogs in an in vitro transcription reaction. The active molecules in the RNA population are selected by an activity assay, and the location of the analog substitution detrimental to activity is identified by cleavage at the phosphorothioate tag with iodine and resolution of the cleavage fragments by gel electrophoresis. This method, which is as easy as RNA sequencing, is applicable to any RNA that can be transcribed in vitro and has an assayable function. Here we describe protocols for the synthesis of phosphorothioate-tagged analogs and their incorporation into RNA transcripts. The incorporation properties and unique biochemical signatures of each individual analog are discussed.

Adenosine↗
PubMed2000

Binding patterns and kinetics of RNase a interaction with RNA.

Kinetics and binding studies of RNase A and its natural polymeric substrate (RNA), as well as the natural mixture of free 3'-ribonucleotides, were performed by difference spectrophotometry. The obtained kinetic saturation curve, with an anomalous nonhyperbolic shape and a distinct transition point, showed the interchange between the two conformational forms of the enzyme. This occurred in a narrow range of substrate concentration. At low substrate concentration, in spite of the existence of one catalytic cleft, RNase A behaves as a cooperative system, perhaps due to the interactions among the four cooperative binding subsites in the active cleft. At high substrate concentration, the conformational change did occur and was accompanied by a decrease in cooperativity and increment of the catalytic constant. The multiphasic shape of the binding curve, which, in the presence of the enzyme, produced 3'-ribonucleotides (as the ligand molecules), shows four binding subsites. The first three subsites are specific for the attachment of phosphate, ribose, and base moieties belonging to the first bound 3'-ribonucleotide in the direction of 3'-phosphate --> ribose --> base-5'. The fourth subsite relates to the second phosphate group of the second bound 3'-ribonucleotide. The binding direction also converts to 5'-phosphate --> ribose --> base-3' for the ribonucleotide monomers in the RNA structure.

Catalysis↗
PubMed1991

Different complexes are formed on the 3' end of histone mRNA with nuclear and polyribosomal proteins.

Specific protein-RNA complexes are formed by incubating a synthetic histone mRNA 3' end (a 30 nucleotide stem-loop structure) RNA with extracts of either nuclei or polyribosomes. The complex formed between the stem-loop and nuclear proteins has a lower electrophoretic mobility than the complex formed between the stem-loop and polyribosomal proteins. Binding of the synthetic 3' end by both polyribosomal and nuclear proteins is abolished when two of the conserved uridine residues in the loop are replaced with adenosines. UV crosslinking of the protein complexes to the synthetic RNA resulted in transferring radiolabel to similar sized proteins, 50 kD, in both the nuclear and polyribosomal extracts.

Animals↗
PubMed2003

RNA as a target for developing antivirals.

The base of knowledge concerning RNA structure and function has been expanding rapidly in recent years. Simultaneously, an increasing awareness of the pivotal role RNA plays in viral diseases has prompted many researchers to apply new technologies in high-throughput screening and molecular modelling to the design of antiviral drugs that target RNA. While the two RNA viruses with the greatest unmet medical need, HIV and HCV, have been most actively pursued, the approaches discussed in this review are relevant to all virus infections. Both traditional small-molecule and large-molecule therapeutics, such as antisense, ribozymes and interfering dsRNAs have been described, and several molecules are under development for commercialization. The purpose of this review is to summarize the current state of the art in this field and to postulate new directions in the future.

Antiviral Agents↗
PubMed2000

ATP can be dispensable for prespliceosome formation in yeast.

The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure.

Adenosine Triphosphate↗
PubMed1996

Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag.

Unspliced cytoplasmic retroviral RNA in chronically infected cells either is encapsidated by Gag proteins in the manufacture of virus or is used to direct synthesis of Gag proteins. Several models have been suggested to explain the sorting of viral RNA for these two purposes. Here we present evidence supporting a simple biochemical mechanism that accounts for the routing of retroviral RNA. Our results indicate that ribosomes compete with the Gag proteins to determine the fate of nascent retroviral RNA. Although the integrity of the entire Rous sarcoma virus leader sequence is important for retroviral packaging and translation, the RNA structure around the third small open reading frame, which neighbors the psi site required for packaging of the RNA, is particularly critical for maintenance of the balance between translation and packaging. These results support the hypothesis that Gag proteins autogenously regulate their synthesis and encapsidation of retroviral RNA and that an equilibrium exists between RNA destined for translation and packaging that is based on the intracellular levels of Gag proteins and ribosomes. To test the model, mRNAs with natural or mutated 5' leader sequences from Rous sarcoma virus were expressed in avian cells in the presence and absence of Pr76gag. We demonstrate that Pr76gag acts as a translational repressor of these mRNAs in a dose-dependent manner, supporting the hypothesis that Pr76gag can sort retroviral RNA for translation and encapsidation.

Animals↗
PubMed2001

Crystal structure of an RNA duplex r(gugucgcac)(2) with uridine bulges.

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gugucgcac)(2), was determined at 1.4 A resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R(work)/R(free) of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4(1)22 with two independent molecules in the asymmetric unit. The unit cell dimensions are a=b=47.18 A and c=80.04 A. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (approximately +50 degrees) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2'-endo conformation while the rest, including the bulges, are in the C3'-endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (approximately 4.4 A) are significantly shorter than the average P-P distance in the duplex (6.0 A). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.

Base Pairing↗
PubMed1998

PRP16, a DEAH-box RNA helicase, is recruited to the spliceosome primarily via its nonconserved N-terminal domain.

Dynamic rearrangement of RNA structure is crucial for intron recognition and formation of the catalytic core during pre-mRNA splicing. Three of the splicing factors that contain sequence motifs characteristic of the DExD/DExH-box family of RNA-dependent ATPases (Prp16, Prp22, and the human homologue of Brr2) recently have been shown to unwind RNA duplexes in vitro, providing biochemical evidence that they may direct structural rearrangements on the spliceosome. Notably, however, the unwinding activity of these proteins is sequence nonspecific, raising the question of how their functional specificity is determined. Because the highly conserved DExD/DExH-box domain in these proteins is typically flanked by one or more nonconserved domains, we have tested the hypothesis that the nonconserved regions of Prp16 determine the functional specificity of the protein. We found that the nonconserved N-terminal domain of Prp16 is (1) essential for viability, (2) required for the nuclear localization of Prp16, and (3) capable of binding to the spliceosome specifically at the step of Prp16 function. Moreover, this domain can interact with the rest of the protein to allow trans-complementation. Based on these results, we propose that the spliceosomal target of the unwinding activity of Prp16, and possibly other DExD/DExH-box splicing factors as well, is defined by factors that specifically interact with the nonconserved domains of the protein.

Adenosine Triphosphatases↗
PubMed2000

Modulation of RNA function by aminoglycoside antibiotics.

One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

Aminoglycosides↗
PubMed2006

Regulatory RNAs as mediators of virulence gene expression in bacteria.

Bacteria exploit functional diversity of RNAs in a wide range of regulatory mechanisms to control gene expression. In last few years, small RNA molecules have been discovered at a staggering rate in bacteria, mainly in Escherichia coli. While functions of many of these RNA molecules are still not known, several of them behave as key effectors of adaptive responses, such as environmental cue recognition, stress response, and virulence control. Most fascinating, perhaps, is the discovery that mRNAs behave as direct sensors of small molecules or of environmental cues. The astonishing diversity of RNA-dependent regulatory mechanisms is linked to the dynamic properties and versatility of the RNA structure. In this review, we relate several recent studies in different bacterial pathogens that illustrate the diverse roles of RNA to control virulence gene expression.

Bacteria↗
PubMed2005

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex.

The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Regulating eukaryotic gene expression with aptamers.

Aptamers are RNA or DNA oligonucleotides identified within a randomly synthesized library, through an in vitro selection procedure. The selected candidates display a pre-determined property of interest with respect to a given target. Successful selection has been carried out against targets ranging from small (amino acids, antibiotics) to macro-molecules (proteins, nucleic acids). They generally show an affinity in the nanomolar range and a high specificity of target recognition. Interestingly, aptamers selected against purified targets in the test tube retain their properties within cells. RNA aptamers can be generated in situ from an appropriate DNA construct or delivered as nuclease-resistant oligonucleotide analogues. For example, aptamers recognizing RNA structure through loop-loop interactions modulate the trans-activation of in vitro transcription mediated by the TAR RNA element of human immunodeficiency virus type 1. Consequently, they constitute both exquisite tools for functional genomics analysis and promising prototypes of therapeutic agents. Natural aptameric motifs have been identified within mRNA sequences, which upon binding to a metabolite control the expression of the encoded gene, which is generally involved in the biosynthesis of this particular metabolite.

Amino Acid Motifs↗
PubMed1993

The type 1 human immunodeficiency virus Tat binding protein is a transcriptional activator belonging to an additional family of evolutionarily conserved genes.

The type 1 human immunodeficiency virus Tat protein is a powerful transcriptional activator when bound to an RNA structure (TAR) present at the extreme 5' terminus of viral mRNA. Since transcriptional activation requires binding of Tat to RNA, it has been suggested that Tat enhances initiation or elongation through a direct interaction with cellular transcription factors. Here we show through protein fusion experiments that the previously identified cellular Tat binding protein, TBP-1, although unable to bind DNA, is a strong transcriptional activator when brought into proximity of several promoter elements. Transcriptional activity depends upon the integrity of at least two highly conserved domains: one resembling a nucleotide-binding motif and the other motif common to proteins with helicase activity. Our studies further reveal that TBP-1 represents one member of a large, highly conserved gene family that encodes proteins demonstrating strong amino acid conservation across species. Finally, we identified a second family member that, although 77% similar to TBP-1, does not activate transcription from the promoters examined. This finding, together with the observation that TBP-1 does not activate each promoter examined, suggests that this gene family may encode promoter-specific transcriptional activators.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2001

Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid.

Potato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates within the nucleus of host cells. To determine how this small, highly structured RNA enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. A cDNA copy of the complete PSTVd genome was, in turn, embedded within the intron, and this construct was delivered into Nicotiana benthamiana plants via a vector based on Potato virus X. The intron-containing GFP subgenomic RNA synthesized during virus infection cannot produce a functional GFP unless the RNA is imported into the nucleus, where the intron can be removed and the spliced RNA returned to the cytoplasm. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear import and RNA splicing events did occur.

Active Transport, Cell Nucleus↗