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 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 Demonstration of Nicking/Joining Activity at the Origin of DNA Replication Associated with the Rep and Rep′ Proteins of Porcine Circovirus Type 1

2006 ◽  
Vol 80 (13) ◽  
pp. 6225-6234 ◽  
Author(s):  
Tobias Steinfeldt ◽  
Tim Finsterbusch ◽  
Annette Mankertz
Keyword(s):  
Dna Replication ◽  
Virus Replication ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Loop Structure ◽  
Stem Loop ◽  
Single Strand Break ◽  
Stem Loop Structure ◽  
Rep Proteins

ABSTRACT The replication of porcine circovirus type 1 (PCV1) is thought to occur by rolling-circle replication (RCR), whereby the introduction of a single-strand break generates a free 3′-hydroxyl group serving as a primer for subsequent DNA synthesis. The covalently closed, single-stranded genome of PCV1 replicates via a double-stranded replicative intermediate, and the two virus-encoded replication-associated proteins Rep and Rep′ have been demonstrated to be necessary for virus replication. However, although postulated to be involved in RCR-based virus replication, the mechanism of action of Rep and Rep′ is as yet unknown. In this study, the ability of PCV1 Rep and Rep′ to “nick” and “join” strand discontinuities within synthetic oligonucleotides corresponding to the origin of replication of PCV1 was investigated in vitro. Both proteins were demonstrated to be able to cleave the viral strand between nucleotides 7 and 8 within the conserved nonanucleotide motif (5′-TAGTATTAC-3′) located at the apex of a putative stem-loop structure. In addition, the Rep and Rep′ proteins of PCV1 were demonstrated to be capable of joining viral single-stranded DNA fragments, suggesting that these proteins also play roles in the termination of virus DNA replication. This joining activity was demonstrated to be strictly dependent on preceding substrate cleavage and the close proximity of origin fragments accomplished by base pairing in the stem-loop structure. The dual “nicking/joining” activities associated with PCV1 Rep and Rep′ are pivotal events underlying the RCR-based replication of porcine circoviruses in mammalian cells.

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 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 Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3′-Terminal Sequences of Viral Positive- and Negative-Strand RNA

2001 ◽  
Vol 75 (4) ◽  
pp. 1708-1721 ◽  
Author(s):  
Rajeev Banerjee ◽  
Asim Dasgupta
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Specific Interaction ◽  
Helicase Domain ◽  
Loop Structure ◽  
Stem Loop ◽  
Negative Strand ◽  
Stem Loop Structure ◽  
Rna Interaction ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ΔpNS3 (helicase domain only) and examined their abilities to interact with the 3′-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3′ untranslated region (UTR). The results presented here demonstrate that NS3 (and ΔpNS3) interacts efficiently and specifically with the 3′-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5′-terminal RNA sequences. The interaction of NS3 with the 3′-terminal negative strand [called 3′(−) UTR127] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3′ terminus (nucleotides 5 to 20 from the 3′ end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ΔpNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3′-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3′(−) UTR binding, specific interaction of NS3 (or ΔpNS3) with the 3′-terminal sequences of the positive-strand RNA [3′(+) UTR] appears to require the entire 3′(+) UTR of HCV. Deletion of either the 98-nucleotide 3′-terminal conserved region or the 5′ half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3′-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

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.

scholarly journals Identification of Tau Stem Loop RNA Stabilizers

2007 ◽  
Vol 12 (6) ◽  
pp. 789-799 ◽  
Author(s):  
Christine P. Donahue ◽  
Jake Ni ◽  
Eriks Rozners ◽  
Marcie A. Glicksman ◽  
Michael S. Wolfe
Keyword(s):  
Small Molecules ◽  
Binding Assay ◽  
Loop Structure ◽  
Stem Loop ◽  
Binding Domains ◽  
Stem Loop Structure ◽  
Exon 10 ◽  
Tau Expression

Alternative splicing of tau exon 10 produces tau isoforms with either 3 (3R) or 4 (4R) repeated microtubule-binding domains. Increased ratios of 4R to 3R tau expression, above the physiological 1:1, leads to neurofibrillary tangles and causes neurodegenerative disease. An RNA stem loop structure plays a significant role in determining the ratio, with decreasing stability correlating with an increase in 4R tau mRNA expression. Recent studies have shown that aminoglycosides are able to bind and stabilize the tau stem loop in vitro, suggesting that other druglike small molecules could be identified and that such molecules might lead to decreased exon 10 splicing in vivo. The authors have developed a fluorescent high-throughput fluorescent binding assay and screened a library of ∼110,000 compounds to identify candidate drugs that will bind the tau stem loop in vitro. In addition, they have developed a fluorescent-based RNA probe to assay the stabilizing effects of candiate drugs on the tau stem loop RNA. These assays should be applicable to the general problem of identifying small molecules that interact with mRNA secondary structures. ( Journal of Biomolecular Screening 2007:789-799)

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