scholarly journals Phosphate Starvation Induces the Sporulation Killing Factor of Bacillus subtilis

2006 ◽  
Vol 188 (14) ◽  
pp. 5299-5303 ◽  
Author(s):  
Nicholas E. E. Allenby ◽  
Carys A. Watts ◽  
Georg Homuth ◽  
Zoltán Prágai ◽  
Anil Wipat ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Intergenic Region ◽  
Phosphate Starvation ◽  
Loop Structure ◽  
Stem Loop ◽  
Specific Transcript ◽  
Stem Loop Structure ◽  
Gel Shift

ABSTRACT Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript.

scholarly journals Archaeal Pyrococcus furiosus thymidylate synthase 1 is an RNA-binding protein

2005 ◽  
Vol 393 (1) ◽  
pp. 373-379 ◽  
Author(s):  
Akio Kanai ◽  
Asako Sato ◽  
Jun Imoto ◽  
Masaru Tomita
Keyword(s):  
Thymidylate Synthase ◽  
Rna Binding ◽  
Pyrococcus Furiosus ◽  
In Vitro Translation ◽  
Luciferase Reporter ◽  
Amino Acid Sequence Similarity ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

Using a stem–loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem–loop structure at a high temperature (75 °C) with an apparent dissociation constant of 0.6 μM. A similar stem–loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem–loop structure. In vitro translation analysis using chimaeric constructs containing the stem–loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem–loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine–Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.

scholarly journals Human Cytomegalovirus UL84 Interacts with an RNA Stem-Loop Sequence Found within the RNA/DNA Hybrid Region of oriLyt

2007 ◽  
Vol 81 (13) ◽  
pp. 7077-7085 ◽  
Author(s):  
Kelly S. Colletti ◽  
Kate E. Smallenburg ◽  
Yiyang Xu ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Specific Interaction ◽  
Simian Virus 40 ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Core Proteins ◽  
Essential Region

ABSTRACT Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex cis-acting oriLyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within oriLyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to oriLyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within oriLyt.

scholarly journals Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

1989 ◽  
Vol 9 (11) ◽  
pp. 5134-5142 ◽  
Author(s):  
M Kozak
Keyword(s):  
Secondary Structure ◽  
Ribosomal Subunit ◽  
Loop Structure ◽  
Entry Site ◽  
Range Interaction ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Delta G

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs

1989 ◽  
Vol 9 (11) ◽  
pp. 5134-5142
Author(s):  
M Kozak
Keyword(s):  
Secondary Structure ◽  
Ribosomal Subunit ◽  
Loop Structure ◽  
Entry Site ◽  
Range Interaction ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Delta G

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

scholarly journals Two Xenopus Proteins That Bind the 3′ End of Histone mRNA: Implications for Translational Control of Histone Synthesis during Oogenesis

1999 ◽  
Vol 19 (1) ◽  
pp. 835-845 ◽  
Author(s):  
Zeng-Feng Wang ◽  
Thomas C. Ingledue ◽  
Zbigniew Dominski ◽  
Ricardo Sanchez ◽  
William F. Marzluff
Keyword(s):  
Translational Control ◽  
Mrna Processing ◽  
Loop Structure ◽  
Histone Mrna ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Stem Loop Structure ◽  
Histone Synthesis

ABSTRACT Translationally inactive histone mRNA is stored in frog oocytes, and translation is activated at oocyte maturation. The replication-dependent histone mRNAs are not polyadenylated and end in a conserved stem-loop structure. There are two proteins (SLBPs) which bind the 3′ end of histone mRNA in frog oocytes. SLBP1 participates in pre-mRNA processing in the nucleus. SLBP2 is oocyte specific, is present in the cytoplasm, and does not support pre-mRNA processing in vivo or in vitro. The stored histone mRNA is bound to SLBP2. As oocytes mature, SLBP2 is degraded and a larger fraction of the histone mRNA is bound to SLBP1. The mechanism of activation of translation of histone mRNAs may involve exchange of SLBPs associated with the 3′ end of histone mRNA.

scholarly journals Replication promiscuity of DNA-β satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-β satellite localizes sequences involved in replication

2008 ◽  
Vol 89 (12) ◽  
pp. 3165-3172 ◽  
Author(s):  
Keith Saunders ◽  
Rob W. Briddon ◽  
John Stanley
Keyword(s):  
Intergenic Region ◽  
Monopartite Begomoviruses ◽  
Yellow Vein ◽  
Cotton Leaf ◽  
Loop Structure ◽  
Origin Of Replication ◽  
Stem Loop ◽  
Deletion Mutagenesis ◽  
Ageratum Yellow Vein Virus ◽  
Stem Loop Structure

Pseudorecombination studies in Nicotiana benthamiana demonstrate that Ageratum yellow vein virus (AYVV) and Eupatorium yellow vein virus (EpYVV) can functionally interact with DNA-β satellites associated with AYVV, EpYVV, cotton leaf curl Multan virus (CLCuMV) and honeysuckle yellow vein virus (HYVV). In contrast, CLCuMV shows some specificity in its ability to interact with distinct satellites and HYVV is able to interact only with its own satellite. Using an N. benthamiana leaf disk assay, we have demonstrated that HYVV is unable to trans-replicate other satellites. To investigate the basis of trans-replication compatibility, deletion mutagenesis of AYVV DNA-β has been used to localize the origin of replication to approximately 360 nt, encompassing the ubiquitous nonanucleotide/stem–loop structure, satellite conserved region (SCR) and part of the intergenic region immediately upstream of the SCR. Additional deletions within this intergenic region have identified a region that is essential for replication. The capacity for DNA-β satellites to functionally interact with distinct geminivirus species and its implications for disease diversification are discussed.

scholarly journals Transcriptional Organization and Posttranscriptional Regulation of the Bacillus subtilis Branched-Chain Amino Acid Biosynthesis Genes

2004 ◽  
Vol 186 (8) ◽  
pp. 2240-2252 ◽  
Author(s):  
Ulrike Mäder ◽  
Susanne Hennig ◽  
Michael Hecker ◽  
Georg Homuth
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Half Life ◽  
Posttranscriptional Regulation ◽  
Full Length ◽  
Loop Structure ◽  
Stem Loop ◽  
Branched Chain Amino Acid ◽  
Stem Loop Structure ◽  
Branched Chain

ABSTRACT In Bacillus subtilis, the genes of the branched-chain amino acids biosynthetic pathway are organized in three genetic loci: the ilvBHC-leuABCD (ilv-leu) operon, ilvA, and ilvD. These genes, as well as ybgE, encoding a branched-chain amino acid aminotransferase, were recently demonstrated to represent direct targets of the global transcriptional regulator CodY. In the present study, the transcriptional organization and posttranscriptional regulation of these genes were analyzed. Whereas ybgE and ilvD are transcribed monocistronically, the ilvA gene forms a bicistronic operon with the downstream located ypmP gene, encoding a protein of unknown function. The ypmP gene is also directly preceded by a promoter sharing the regulatory pattern of the ilvA promoter. The ilv-leu operon revealed complex posttranscriptional regulation: three mRNA species of 8.5, 5.8, and 1.2 kb were detected. Among them, the 8.5-kb full-length primary transcript exhibits the shortest half-life (1.2 min). Endoribonucleolytic cleavage of this transcript generates the 5.8-kb mRNA, which lacks the coding sequences of the first two genes of the operon and is predicted to carry a stem-loop structure at its 5′ end. This processing product has a significantly longer half-life (3 min) than the full-length precursor. The most stable transcript (half-life, 7.6 min) is the 1.2-kb mRNA generated by the processing event and exonucleolytic degradation of the large transcripts or partial transcriptional termination. This mRNA, which encompasses exclusively the ilvC coding sequence, is predicted to carry a further stable stem-loop structure at its 3′ end. The very different steady-state amounts of mRNA resulting from their different stabilities are also reflected at the protein level: proteome studies revealed that the cellular amount of IlvC protein is 10-fold greater than that of the other proteins encoded by the ilv-leu operon. Therefore, differential segmental stability resulting from mRNA processing ensures the fine-tuning of the expression of the individual genes of the operon.

Mechanism of puf mRNA degradation: the role of an intercistronic stem-loop structure

Gene ◽  
1988 ◽  
Vol 72 (1-2) ◽  
pp. 109-117 ◽  
Author(s):  
Joel G. Belasco ◽  
Chyi-Ying A. Chen
Keyword(s):  
Mrna Degradation ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

scholarly journals Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

1999 ◽  
Vol 19 (6) ◽  
pp. 4334-4342 ◽  
Author(s):  
B. Stefanovic ◽  
C. Hellerbrand ◽  
D. A. Brenner
Keyword(s):  
Protein Complexes ◽  
Stellate Cell ◽  
State Level ◽  
Start Codon ◽  
Loop Structure ◽  
Fibrillar Collagen ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Reporter Mrna

ABSTRACT Three fibrillar collagen mRNAs, α1(I), α2(I), and α1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen α1(I) reporter mRNAs in hsc’s and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc’s and to a lesser extent in activated hsc’s and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc’s and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3′ untranslated region (UTR) of collagen α1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc’s. This 3′ UTR binds αCP, a protein implicated in increasing stability of collagen α1(I) mRNA in activated hsc’s (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201–5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen α1(I) mRNA is regulated by a complex interaction between the 5′ stem-loop and the 3′ UTR, which may optimize collagen production in activated hsc’s.

scholarly journals In Vitro Synthesis of Minus-Strand RNA by an Isolated Cereal Yellow Dwarf Virus RNA-Dependent RNA Polymerase Requires VPg and a Stem-Loop Structure at the 3′ End of the Virus RNA

2006 ◽  
Vol 80 (21) ◽  
pp. 10743-10751 ◽  
Author(s):  
Toba A. M. Osman ◽  
Robert H. A. Coutts ◽  
Kenneth W. Buck
Keyword(s):  
Rna Polymerase ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Loop Structure ◽  
Minus Strand ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Virus Rna ◽  
Yellow Dwarf Virus

ABSTRACT Cereal yellow dwarf virus (CYDV) RNA has a 5′-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3′-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3′ terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3′ end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3′-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

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