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At least 19 recordsLinked to original sources
PubMed1994

The regulatory RNA gene micF is present in several species of gram-negative bacteria and is phylogenetically conserved.

micF RNA post-transcriptionally regulates Escherichia coli outer membrane protein F (OmpF), in response to temperature increase and other environmental stress conditions, by binding to ompF mRNA and destabilizing the message. Southern analyses show that the micF gene is present in related Gram-negative bacteria, including Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In addition, Northern analyses indicate that micF RNA and ompF mRNA levels are thermally regulated in several related species in a manner similar to the thermoregulation in Escherichia coli. DNA sequences from Salmonella typhi, Salmonella typhimurium, and Klebsiella pneumoniae show greater than 96% homology in the micF gene when compared to the Escherichia coli micF sequence. Upstream of micF, sequences show considerable variation, although several distinct regions are highly conserved. Some of these conserved regions correspond to known binding sites for the transcription factor OmpR and the DNA-binding protein integration host factor. In addition, E. coli micF RNA incubated with protein extracts from other species forms heterologous ribonucleoproteins (RNPs). The formation of these heterologous RNPs indicates both the presence of micF RNA-binding protein homologues in other species and a conservation of RNA-protein recognition sites. This work demonstrates that the micF RNA regulatory system is present in other Gram-negative bacterial species and that this system appears to be phylogenetically conserved.

Bacterial Outer Membrane Proteins↗
PubMed1995

Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa: kinship to coliphages and conservation of regulatory RNA structures.

We report the complete nucleotide sequence of the single-stranded RNA phage PP7 from Pseudomonas aeruginosa. There are three open reading frames which code for apparent protein homologues of the single-stranded RNA coliphages, i.e., maturation protein, coat protein, and replicase. A fourth overlapping reading frame exists that probably encodes a lysis protein, similar to what has been found in the group A coliphages such as MS2. The genetic map of PP7 is colinear with group A coliphages and we accordingly classify the phage as a levivirus. There is, generally speaking, no significant nucleotide sequence identity between PP7 and the coliphages except for a few regions where homologous parts of proteins are encoded, most notable in the replicase gene. In these regions the nucleotide sequence similarity between PP7 and MS2 is no greater than between PP7 and the group B coliphages such as Q beta. Surprisingly, Q beta and MS2 are no closer to each other than they are to PP7. Several regulatory RNA secondary structure features that are present in the coliphages were identified also in PP7 RNA although the sequences involved cannot be aligned. Among these are the coat protein binding helix at the start of the replicase gene, structures at the 5' and 3' terminus of the RNA, a replicase binding site, and the structure of the coat protein cistron start. Some of these features resemble MS2 type coliphages but others the Q beta type. These findings suggest that PP7 is related to the coliphages but branched off before the coliphages diverged into separate groups.

Amino Acid Sequence↗
PubMed1995

Polypyrimidine tract-binding protein and heterogeneous nuclear ribonucleoprotein A1 bind to human T-cell leukemia virus type 2 RNA regulatory elements.

Efficient expression of human T-cell leukemia virus (HTLV) and human immunodeficiency virus structural proteins requires Rx and Rev proteins, respectively. Decreased expression of Gag and Env appears to be due, in part, to intragenic RNA sequences, termed cis-acting repressive sequences (CRS), and may be mediated by binding of specific cellular factors. We demonstrated previously that two cellular proteins, p60CRS and p40CRS, interact with HTLV type 2.5' long terminal repeat CRS RNA and that the interaction of both proteins with CRS RNA correlates with function (A. C. Black, C. T. Ruland, J. Luo, A. Bakker, J. K. Fraser, and J. D. Rosenblatt, Virology 200:29-41, 1994). By radioimmunoprecipitation of HeLa nuclear proteins UV cross-linked to CRS RNAs with murine monoclonal antibodies, we now show that p40CRS is heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and p60CRS is polypyrimidine tract-binding protein or hnRNP I. These immunoprecipitation results were confirmed by an immunobinding assay with hnRNP I and hnRNP AI antibodies and by cross-competition electrophoretic mobility shift experiments. In addition, we mapped a putative hnRNP A1 binding site in U5 RNA and demonstrated that p40CRS (hnRNP A1) binding to that site correlates with CRS function. Since both hnRNP I and hnRNP A1 have been shown to influence splicing and potentially other steps in RNA processing, the binding of both hnRNP I and hnRNP A1 to HTLV RNA regulatory elements may alter retrovirus RNA processing and may be involved in regulation by Rex.

Base Sequence↗
PubMed1993

Multiple RNA regulatory elements mediate distinct steps in localization of oskar mRNA.

Pattern formation in the early development of many organisms relies on localized cytoplasmic proteins, which can be prelocalized as mRNAs. The Drosophila oskar gene, required both for posterior body patterning and germ cell determination, encodes one such mRNA. Localization of oskar mRNA is an elaborate process involving movement of the transcript first into the oocyte from adjacent interconnected nurse cells and then across the length of the oocyte to its posterior pole. We have mapped RNA regulatory elements that direct this localization. Using a hybrid lacZ/oskar mRNA, we identify several elements within the oskar 3' untranslated region that affect different steps in the process: the early movement into the oocyte, accumulation at the anterior margin of the oocyte and finally localization to the posterior pole. This use of multiple cis-acting elements suggests that localization may be orchestrated in a combinatorial fashion, thereby allowing localized mRNAs with ultimately different destinations to employ common mechanisms for shared intermediate steps.

Animals↗
PubMed1997

Forced evolution of a regulatory RNA helix in the HIV-1 genome.

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.

Base Sequence↗
PubMed1987

S4-alpha mRNA translation regulation complex. II. Secondary structures of the RNA regulatory site in the presence and absence of S4.

The secondary structure of the Escherichia coli alpha mRNA leader sequence has been determined using nucleases specific for single- or double-stranded RNA. Three different length alpha RNA fragments were studied at 0 degrees C and 37 degrees C. A very stable eight base-pair helix forms upstream from the ribosome initiation site, defining a 29 base loop. There is evidence for base-pairing between nucleotides within this loop and for a "pseudo-knot" interaction of some loop bases with nucleotides just 3' to the initiation codon, forming a region of complex structure. A weak helix also pairs sequences near the 5' terminus of the alpha mRNA with bases near the Shine-Dalgarno sequence. Affinity constants for the translational repressor S4 binding different length alpha mRNA fragments indicate that most of the S4 recognition features must be contained within the main helix and hairpin regions. Binding of S4 to the alpha mRNA alters the structure of the 29 base hairpin region, and probably melts the weak pairing between the 5' and 3' termini of the leader. The pseudo-knot structure and the conformational changes associated with it provide a link between the structures of the S4 binding site and the ribosome binding site. The alpha mRNA may therefore play an active role in mediating translational repression.

Autoradiography↗
PubMed1993

Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule.

The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up-regulated by agr whereas surface proteins are down-regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr-null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

Base Sequence↗
PubMed1991

RNA regulatory elements mediate control of Drosophila body pattern by the posterior morphogen nanos.

In Drosophila embryos, graded activity of the posterior determinant nanos (nos) generates abdominal segmentation by blocking protein expression from maternal transcripts of the hunchback (hb) gene. When active inappropriately at the anterior pole, nos can also block expression of the anterior determinant bicoid (bcd). We show that both regulatory interactions are mediated by similar sequences in the 3' untranslated region of each transcript. These nos response elements (NREs) are both necessary and sufficient to confer nos-dependent regulation, the degree of regulation determined by the number and quality of the elements and the level of nos in vivo. Based on these and other results, we argue that nos acts as a morphogen, controlling hb expression (and hence abdominal pattern) as a function of its concentration-dependent interaction with the NREs.

Animals↗
PubMed1988

Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation.

The biosynthetic rates for both the transferrin receptor (TfR) and ferritin are regulated by iron. An iron-responsive element (IRE) in the 5' untranslated portion of the ferritin messenger RNA (mRNA) mediates iron-dependent control of its translation. In this report the 3' untranslated region of the mRNA for the human TfR was shown to be necessary and sufficient for iron-dependent control of mRNA levels. Deletion studies identified a 678-nucleotide fragment of the TfR complementary DNA that is critical for this iron regulation. Five potential stem-loops that resemble the ferritin IRE are contained within the region critical for TfR regulation. Each of two of the five TfR elements was independently inserted into the 5' untranslated region of an indicator gene transcript. In this location they conferred iron regulation of translation. Thus, an mRNA element has been implicated in the mediation of distinct regulatory phenomena dependent on the context of the element within the transcript.

Animals↗
PubMed1990

Cloning of the cDNA encoding an RNA regulatory protein--the human iron-responsive element-binding protein.

Iron-responsive elements (IREs) are stemloop structures found in the mRNAs encoding ferritin and the transferrin receptor. These elements participate in the iron-induced regulation of the translation of ferritin and the stability of the transferrin receptor mRNA. Regulation in both instances is mediated by binding of a cytosolic protein to the IREs. High-affinity binding is seen when cells are starved of iron and results in repression of ferritin translation and inhibition of transferrin receptor mRNA degradation. The IRE-binding protein (IRE-BP) has been identified as an approximately 90-kDa protein that has been purified by both affinity and conventional chromatography. In this report we use RNA affinity chromatography and two-dimensional gel electrophoresis to isolate the IRE-BP for protein sequencing. A degenerate oligonucleotide probe derived from a single peptide sequence was used to isolate a cDNA clone that encodes a protein containing 13 other sequenced peptides obtained from the IRE-BP. Consistent with previous characterization of the IRE-BP, the cDNA encodes a protein of 87 kDa with a slightly acidic pI, and the corresponding mRNA of approximately 3.6 kilobases is found in a variety of cell types. The encoded protein contains a nucleotide-binding consensus sequence and regions of cysteine and histidine clusters. This mRNA is encoded by a single gene on human chromosome 9, a finding consistent with previous localization by functional mapping. The protein contains no previously defined consensus motifs for either RNA or DNA binding. The simultaneous cloning of a different, but highly homologous, cDNA suggests that the IRE-BP is a member of a distinct gene family.

Amino Acid Sequence↗
PubMed1993

Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid.

The efficiency of conjugation of F-like plasmids is regulated by the FinOP fertility inhibition system. The transfer (tra) operon is under the direct control of the TraJ transcriptional activator which, in turn, is negatively regulated by FinP, an antisense RNA, and FinO, a 22 kDa protein. Recently, FinO has been shown to extend the chemical stability of FinP in vivo in the absence of traJ mRNA. The in vitro secondary structures of both the FinP and TraJ RNAs were determined by the use of single- and double-strand-specific nucleases; both RNAs were found to have double stem-loop structures that are complementary to each other and, therefore, FinP RNA and TraJ RNA have the potential to form a duplex with each other. This was verified by in vitro binding experiments. The reaction was shown to be biomolecular with an apparent rate constant (kapp) of 5 x 10(5)M-1s-1, a value that is similar to those found for other natural antisense RNA systems. Preliminary evidence for the in vivo formation of the FinP-TraJ RNA duplex was obtained by primer extension of the traJ mRNA; the presence of both FinO and FinP was required to cause a dramatic reduction in the steady-state level of traJ mRNA, perhaps as a result of RNase III degradation of the resulting RNA duplex.

Bacterial Outer Membrane Proteins↗
PubMed1993

RNA regulatory element BLE1 directs the early steps of bicoid mRNA localization.

Deployment of the bicoid morphogen gradient in early Drosophila embryos requires the prelocalization of bicoid mRNA to the anterior pole of the egg. This anterior localization is mediated by a cis-acting localization signal contained within the 3' untranslated region of the bicoid mRNA. Here we use a series of bicoid transgenes carrying small deletions in the 3' untranslated region to survey for functional elements that constitute the localization signal. We identify and characterize one essential element, BLE1, which specifically directs the early steps of localization. In addition, we find that many deletions within the bicoid mRNA 3' untranslated region impair but do not prevent localization. One such deletion specifically interferes with a later step in localization. Thus the bicoid mRNA localization signal appears to consist of multiple different elements, each responsible for different steps in the localization process.

Animals↗
PubMed1994

Leeway and constraints in the forced evolution of a regulatory RNA helix.

The start of the coat protein gene of RNA phage MS2 adopts a well-defined hairpin structure of 12 bp (including one mismatch) in which the start codon occupies the loop position. An earlier expression study using partial MS2 cDNA clones had indicated that the stability of this hairpin is important for gene expression. For every -1.4 kcal/mol increase in stability a 10-fold reduction in coat protein was obtained. Destabilizations beyond the wild-type value did not affect expression. These results suggested that the hairpin was tuned in the sense that it has the highest stability still compatible with maximal ribosome loading. Employing an infectious MS2 cDNA clone, we have now tested the prediction that the delta G 0 of the coat protein initiator helix is set at a precise value. We have introduced stabilizing and destabilizing mutations into this hairpin in the intact phage and monitored their evolution to viable species. By compensatory mutations, both types of mutants quickly revert along various pathways to wild-type stability, but not to wild-type sequence. As a rule the second-site mutations do not change the encoded amino acids or the Shine-Dalgarno sequence. The return of too strong hairpins to wild-type stability can be understood from the need to produce adequate supplies of coat protein. The return of unstable hairpins to wild-type stability is not self-evident and is presently not understood. The revertants provide an evolutionary landscape of slightly suboptimal phages, that were stable at least for the duration of the experiment (approximately 20 infection cycles).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1995

Translation of RNAIII, the Staphylococcus aureus agr regulatory RNA molecule, can be activated by a 3'-end deletion.

RNAIII, an RNA molecule shown to encode delta-hemolysin and independently to regulate toxin synthesis in Staphylococcus aureus, is transcribed at the mid-exponential phase of growth, while its target genes are activated 2 h later, at the post-exponential phase of growth. We show here that the translation of RNAIII to the 26-amino acid peptide delta-hemolysin is delayed by 1 h, and that this delay is abolished when the 3'-end of this molecule is deleted. We suggest that structural changes of RNAIII to a translatable form of the molecule precede its regulation of target gene expression.

Bacterial Proteins↗
PubMed1991

Homology between IRE-BP, a regulatory RNA-binding protein, aconitase, and isopropylmalate isomerase.

Iron-responsive elements (IREs) are regulatory RNA elements which serve as specific binding sites for the IRE-binding protein (IRE-BP). Interaction between IREs and IRE-BP induces repression of ferritin mRNA translation and transferrin receptor mRNA stabilization. We describe the identification of extensive amino acid sequence homology between IRE-BP and two known isomerases, aconitase and isopropylmalate (IPM) isomerase. We discuss the implications of this observation with regard to structure/function relationships of IRE-BP. The structural conservation between a regulatory RNA-binding protein and two enzymes involved in intermediary metabolism provides a surprising example of the functional flexibility in biological structures.

Aconitate Hydratase↗
PubMed1995

Testis-brain RNA-binding protein, a testicular translational regulatory RNA-binding protein, is present in the brain and binds to the 3' untranslated regions of transported brain mRNAs.

Previous studies have demonstrated that a phosphoprotein in testis binds to transcript c, a sequence containing two highly conserved elements, Y and H, in the 3' untranslated region (UTR) of mouse protamine 2 mRNA (mP2) and represses its translation in vitro. When gel-retardation assays were performed with cytoplasmic extracts prepared from seven different mouse tissues, we found that brain in addition to testis contains a protein that binds to transcript c. Both the testis and brain proteins are found exclusively in the nonpolysomal fractions of their postmitochondrial extracts. The testis and brain proteins appear to be identical according to numerous criteria: the complexes they form with transcript c have identical mobility in native gels, identical optimal pH, identical lability to increased salt concentrations, identical chromatographic properties, identical molecular sizes as judged from UV crosslinking, and identical peptide mapping as revealed by V8 digestion of the UV crosslinked protein-RNA complexes. In addition to binding to the same conserved sequence in the 3'UTR of mP2, the phosphoprotein from testis and brain, hereafter called testis-brain RNA-binding protein (TB-RBP), also specifically binds to a similar sequence in the 3'UTR of brain Tau mRNA. Since TB-RBP binds to the 3'UTRs of several translationally regulated mRNAs in testis and since numerous transported brain mRNAs also contain the same conserved binding elements, we propose that TB-RBP plays a role in mRNA storage, translocation, and/or localization in brain and testis.

Ammonium Sulfate↗