scholarly journals Identification of a Residue (Glu60) in TRAP Required for Inducing Efficient Transcription Termination at the trp Attenuator Independent of Binding Tryptophan and RNA

2017 ◽  
Vol 199 (6) ◽  
Author(s):  
Natalie M. McAdams ◽  
Andrea Patterson ◽  
Paul Gollnick
Keyword(s):  
Rna Binding ◽  
Transcription Termination ◽  
Genetic Selection ◽  
Leader Region ◽  
Content Type ◽  
Attenuation Mechanism ◽  
Attenuation Models ◽  
A Site ◽  
Additional Role

ABSTRACT Transcription of the tryptophan (trp) operon in Bacillus subtilis is regulated by an attenuation mechanism. Attenuation is controlled by the t rp RNA-binding attenuation protein (TRAP). TRAP binds to a site in the 5′ leader region of the nascent trp transcript in response to the presence of excess intracellular tryptophan. This binding induces transcription termination upstream of the structural genes of the operon. In prior attenuation models, the role of TRAP was only to alter the secondary structure of the leader region RNA so as to promote formation of the trp attenuator, which was presumed to function as an intrinsic terminator. However, formation of the attenuator alone has been shown to be insufficient to induce efficient termination, indicating that TRAP plays an additional role in this process. To further examine the function of TRAP, we performed a genetic selection for mutant TRAPs that bind tryptophan and RNA but show diminished termination at the trp attenuator. Five such TRAP mutants were obtained. Four of these have substitutions at Glu60, three of which are Lys (E60K) substitutions and the fourth of which is a Val (E60V) substitution. The fifth mutant obtained contains a substitution at Ile63, which is on the same β-strand of TRAP as Glu60. Purified E60K TRAP binds tryptophan and RNA with properties similar to those of the wild type but is defective at inducing termination at the trp attenuator in vitro. IMPORTANCE Prior models for attenuation control of the B. subtilis trp operon suggested that the only role for TRAP is to bind to the leader region RNA and alter its folding to induce formation of an intrinsic terminator. However, several recent studies suggested that TRAP plays an additional role in the termination mechanism. We hypothesized that this function could involve residues in TRAP other than those required to bind tryptophan and RNA. Here we obtained TRAP mutants with alterations at Glu60 that are deficient at inducing termination in the leader region while maintaining tryptophan and RNA binding properties similar to those of the WT protein. These studies provide additional evidence that TRAP-mediated transcription termination at the trp attenuator is neither intrinsic nor Rho dependent.

Transcription termination at the chicken beta H-globin gene

1988 ◽  
Vol 8 (12) ◽  
pp. 5369-5377
Author(s):  
T M Pribyl ◽  
H G Martinson
Keyword(s):  
Rna Polymerase Ii ◽  
Inverted Repeat ◽  
Transcription Termination ◽  
Globin Gene ◽  
H Gene ◽  
Delta G ◽  
A Site ◽  
Cat Expression

We characterized the transcription termination region of the chicken beta H-globin gene. First we located the region by nuclear runon transcription in vitro. Then we sequenced and subcloned it into a chloramphenicol acetyltransferase (CAT) expression vector for assay in vivo. The region of beta H termination contains two interesting elements located about 1 kilobase downstream of the beta H gene poly(A) site. Either element alone can block CAT expression if inserted between the promoter and the poly(A) site of the cat gene in pRSVcat. The first element in the termination region is an unusually large inverted repeat in the DNA (delta G = -71 kcal). The second element, 200 base pairs further downstream, is an RNA polymerase II promoter which directs transcription back upstream on the complementary strand. This transcription converges on and collides with that from the beta H gene at or near the inverted repeat where transcription from both directions stops. We propose that the inverted repeat is a strong pause site which positions the converging polymerases for mutual site-specific termination.

scholarly journals Effects of Bicyclomycin on RNA- and ATP-Binding Activities of Transcription Termination Factor Rho

1998 ◽  
Vol 42 (3) ◽  
pp. 571-578 ◽  
Author(s):  
Lucia Carrano ◽  
Cecilia Bucci ◽  
Roberto De Pascalis ◽  
Alfredo Lavitola ◽  
Filomena Manna ◽  
...  
Keyword(s):  
Rna Binding ◽  
Transcription Termination ◽  
Gel Filtration ◽  
Experimental System ◽  
Cross Linking ◽  
Atp Binding ◽  
Mobility Shift ◽  
Termination Factor ◽  
Transcription Termination Factor

ABSTRACT Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within thehisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

scholarly journals Regulation of PACT-Mediated Protein Kinase Activation by the OV20.0 Protein of Orf Virus

2015 ◽  
Vol 89 (22) ◽  
pp. 11619-11629 ◽  
Author(s):  
Yeu-Yang Tseng ◽  
Guan-Ru Liao ◽  
Ganes C. Sen ◽  
Fong-Yuan Lin ◽  
Wei-Li Hsu
Keyword(s):  
Protein Kinase ◽  
Rna Binding ◽  
Small Ruminants ◽  
Kinase Domain ◽  
Orf Virus ◽  
Infection Model ◽  
Content Type ◽  
Binding Domains ◽  
Mouse Infection Model

ABSTRACTDouble-stranded RNA (dsRNA)-activated protein kinase (PKR), a major component of the cellular antiviral system, is activated by the binding of either dsRNA or the cellular PKR activator, the PACT protein. The suppression of PKR activation is one of the main strategies that viruses employ to circumvent interferon signaling. Orf virus (ORFV), a parapoxvirus from thePoxviridaefamily, causes contagious pustular dermatitis in small ruminants. Previous studies have demonstrated that various OV20.0 isoforms, encoded by the OV20.0L gene, are able to inhibit PKR activation both by sequestering dsRNA and by physically interacting with PKRin vitro. Thus, this gene acts as a virulence factor of ORFV when tested using a mouse infection model. In the present study, the regions within OV20.0 that interact with dsRNA and with PKR have been mapped. Furthermore, this study demonstrates for the first time that OV20.0 is also able to interact with the dsRNA binding domain of PACT and that the presence of dsRNA strengthened the interaction of these two molecules. The presence of OV20.0 diminishes PKR phosphorylation when this is stimulated by PACT. Nevertheless, the association of OV20.0 with PKR, rather than with PACT, was found to be essential for reducing PACT-mediated PKR phosphorylation. These observations elucidate a new strategy whereby innate immunity can be evaded by ORFV.IMPORTANCEOur previous study indicated that ORFV's two OV20.0 isoforms act as a PKR antagonist via sequestering the PKR activator, dsRNA, and by interacting with PKR, leading to an inhibition of PKR activation (Y. Y. Tseng, F. Y. Lin, S. F. Cheng, D. Tscharke, S. Chulakasian, C. C. Chou, Y. F. Liu, W. S. Chang, M. L. Wong, and W. L. Hsu, J Virol89:4966–4979, 2015, doi:10.1128/JVI.03714-14). In the current study, the possible mechanisms by which OV20.0 protein counteracts PKR activation were studied in depth. OV20.0 is able to bind PKR and its two activators, dsRNA and PACT. In addition, OV20.0 binds directly to the RNA binding domains (RBDs) of PKR, and this interaction does not require dsRNA. Moreover, OV20.0 interacts with or occupies the RBD2 and the kinase domain of PKR, which then prevents PACT binding to PKR. Finally, OV20.0 associates with PACT via the RBDs, which may reduce the ability of PACT to induce PKR activation. The findings in this study provide new concepts in relation to how ORFV modulates PKR activation.

scholarly journals SR-Like RNA-Binding Protein Slr1 Affects Candida albicans Filamentation and Virulence

2013 ◽  
Vol 81 (4) ◽  
pp. 1267-1276 ◽  
Author(s):  
Chaiyaboot Ariyachet ◽  
Norma V. Solis ◽  
Yaoping Liu ◽  
Nemani V. Prasadarao ◽  
Scott G. Filler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Candida Albicans ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Umbilical Vein ◽  
Polarized Growth ◽  
Content Type ◽  
Fungal Burden

ABSTRACTCandida albicanscauses both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of bothSaccharomyces cerevisiaeandAspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence ofC. albicans. BLAST searches withS. cerevisiaeSR-like protein Npl3 (ScNpl3) identified twoC. albicansproteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, anotherSR-likeRNA-binding protein with no closeS. cerevisiaeortholog. Whereas ScNpl3 was critical for growth, deletion ofNPL3inC. albicansresulted in few phenotypic changes. In contrast, theslr1Δ/Δ mutant had a reduced growth ratein vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cellsin vitro. Mice infected intravenously with theslr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type orslr1Δ/Δ mutant complemented withSLR1(slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization ofslr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected withslr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cellsin vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin onslr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influencesC. albicansgrowth, filamentation, host cell interactions, and virulence.

scholarly journals A 5′ RNA Stem-Loop Participates in the Transcription Attenuation Mechanism That Controls Expression of theBacillus subtilis trpEDCFBA Operon

1999 ◽  
Vol 181 (18) ◽  
pp. 5742-5749 ◽  
Author(s):  
Subita Sudershana ◽  
Hansen Du ◽  
Madhumita Mahalanabis ◽  
Paul Babitzke
Keyword(s):  
Rna Binding ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Regulatory Function ◽  
Structural Genes ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Attenuation Mechanism ◽  
Transcription Attenuation

ABSTRACT The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBAoperon by transcription attenuation. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats. TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach the trp structural genes. In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5′ end of the trp leader transcript. Deletion of this structure resulted in a dramatic increase in expression of atrpE′-′lacZ translational fusion and a reduced ability to regulate expression in response to tryptophan. By introducing a series of point mutations in the 5′ stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels. Our results indicate that the 5′ stem-loop functions primarily through the TRAP-dependent regulatory pathway. Gel shift results demonstrate that the 5′ stem-loop increases the affinity of TRAP for trpleader RNA four- to fivefold, suggesting that the 5′ structure interacts with TRAP. In vitro transcription results indicate that this 5′ structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough. An oligonucleotide complementary to a segment of the 5′ stem-loop was used to demonstrate that formation of the 5′ structure is required for proper attenuation control of this operon.

Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites

1993 ◽  
Vol 13 (9) ◽  
pp. 5159-5167
Author(s):  
L E Hyman ◽  
C L Moore
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
In Vitro Synthesis ◽  
Polyadenylation Sites ◽  
Polymerase Pausing ◽  
A Site ◽  
Pause Site

Little is known about the transcriptional events which occur downstream of polyadenylation sites. Although the polyadenylation site of a gene can be easily identified, it has been difficult to determine the site of transcription termination in vivo because of the rapid processing of pre-mRNAs. Using an in vitro approach, we have shown that sequences from the 3' ends of two different Saccharomyces cerevisiae genes, ADH2 and GAL7, direct transcription termination and/or polymerase pausing in yeast nuclear extracts. In the case of the ADH2 sequence, the RNA synthesized in vitro ends approximately 50 to 150 nucleotides downstream of the poly(A) site. This RNA is not polyadenylated and may represent the primary transcript. A similarly sized nonpolyadenylated [poly(A)-] transcript can be detected in vivo from the same transcriptional template. A GAL7 template also directs the in vitro synthesis of an RNA which extends a short distance past the poly(A) site. However, a significant amount of the GAL7 RNA is polyadenylated at or close to the in vivo poly(A) site. Mutations of GAL7 or ADH2 poly(A) signals prevent polyadenylation but do not affect the in vitro synthesis of the extended poly(A)- transcript. Since transcription of the mutant template continues through this region in vivo, it is likely that a strong RNA polymerase II pause site lies within the 3'-end sequences. Our data support the hypothesis that the coupling of this pause site to a functional polyadenylation signal results in transcription termination.

scholarly journals An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

2014 ◽  
Vol 14 (2) ◽  
pp. 149-157 ◽  
Author(s):  
Natalie M. McAdams ◽  
Michelle L. Ammerman ◽  
Julee Nanduri ◽  
Kaylen Lott ◽  
John C. Fisk ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Mitochondrial Rna ◽  
Insect Vector ◽  
Sequence Specificity ◽  
Content Type

ABSTRACT In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo , inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei . TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

scholarly journals Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Sameer Dixit ◽  
Michaela Müller-McNicoll ◽  
Vojtěch David ◽  
Kathi Zarnack ◽  
Jernej Ule ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Affinity Purification ◽  
Mitochondrial Rna ◽  
Nucleotide Resolution ◽  
Additional Role

ABSTRACT A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei . Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1). Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP) revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome. IMPORTANCE Trypanosoma brucei mitochondrial mRNAs undergo maturation by RNA editing, a unique process involving decrypting open reading frames by the precise deletion and/or insertion of uridine (U) residues at specific positions on an mRNA. This process is catalyzed by multiprotein complexes, such as the RNA editing core complex, which provides the enzymatic activities needed for U insertion/deletion at a single editing site. Less well understood is how RNA editing occurs throughout an mRNA bearing multiple sites. To address this question, we mapped at single-nucleotide resolution the RNA interactions of two unique RNA-binding proteins (RBPs). These RBPs are part of the mitochondrial RNA-binding complex 1, hypothesized to mediate multiple rounds of RNA editing. Both RBPs were shown to mark mRNAs for the process in correlation with the number of editing sites on the transcript. Surprisingly, one also binds mRNAs that bypass RNA editing, indicating that it may have an additional role outside RNA editing.

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