Transcription Antitermination Protein Research Articles

Identification of potential Campylobacter jejuni genes involved in biofilm formation by EZ-Tn5 Transposome mutagenesis

BackgroundBiofilm formation has been suggested to play a role in the survival of Campylobacter jejuni in the environment and contribute to the high incidence of human campylobacteriosis. Molecular studies of biofilm formation by Campylobacter are sparse.ResultsWe attempted to identify genes that may be involved in biofilm formation in seven C. jejuni strains through construction of mutants using the EZ-Tn5 Transposome system. Only 14 mutants with reduced biofilm formation were obtained, all from one strain of C. jejuni. Three different genes of interest, namely CmeB (synthesis of multidrug efflux system transporter proteins), NusG (transcription termination and anti-termination protein) and a putative transmembrane protein (involved in membrane protein function) were identified. The efficiency of the EZ::TN5 transposon mutagenesis approach was strain dependent and was unable to generate any mutants from most of the strains used.ConclusionsA diverse range of genes may be involved in biofilm formation by C. jejuni. The application of the EZ::TN5 system for construction of mutants in different Campylobacter strains is limited.

Proteomics of <i>E.coli Nissle 1917</i> in Responce to<i> Cocos nucifera</i> sap and Wine

In the present study, we described the protein profile experimentally by 2D-PAGE and MALDI analysis to understand the stress mechanisms of cocoti sap and wine on E.coli Nissle 1917. We isolated one newly expressed protein from cocoti wine treated gel which is not present in both control and cocoti sap treated sample i.e. P21 prophage-derived head-stabilizing proteinVG03_ECOL6 (3n1) also called as Head protein gp3. This protein mainly activities related to the viral life cycle. It helps to attach the viral gene into host. The growth rate was delayed in cocoti wine treated E.coli Nissle 1917 when compared to control and cocoti sap treated samples. Stress mechanism induce many proteins they are involved in metabolic process, hydrolase activity, lyase activity, quinone binding, phosphotransferase system, carbohydrate metabolism, DNA binding, DNA repair, transferase activity, oxidoreductase, purine metabolism, transcription anti-termination, transcription regulation and other related activities.We proved that the predicted protein structure quality, resolution, density and error plot values by QMEAN analysis. Based on these results, only two differentially expressed proteins under sap stress showed that the significant results, which were N-acetylgalactosamine-specific phosphotransferase enzyme IIB component 1, PTPB1_ECOLI and DinI-like protein Z3305/ECs2939 in prophage CP-933VDINI1_ECO57. In case of wine stress, the differentially expressed proteins were Transcription anti-termination protein RFAH- ECO57 NusA and PUR7-eco24- phosphoribosylamidazole-succinocarboxamide synthase showed significant results. ProtParam analysis indicating that the multiple physico-chemical characters of differentially expressed proteins were differed and compared. The phylogenetic tree represents the relationship in-between the differentially expressed proteins, were showed siblings (related) as well as monophytic clade.

Proteomics of <i>E.coli Nissle 1917</i> in Responce to<i> Cocos nucifera</i> sap and Wine

In the present study, we described the protein profile experimentally by 2D-PAGE and MALDI analysis to understand the stress mechanisms of cocoti sap and wine on E.coli Nissle 1917. We isolated one newly expressed protein from cocoti wine treated gel which is not present in both control and cocoti sap treated sample i.e. P21 prophage-derived head-stabilizing proteinVG03_ECOL6 (3n1) also called as Head protein gp3. This protein mainly activities related to the viral life cycle. It helps to attach the viral gene into host. The growth rate was delayed in cocoti wine treated E.coli Nissle 1917 when compared to control and cocoti sap treated samples. Stress mechanism induce many proteins they are involved in metabolic process, hydrolase activity, lyase activity, quinone binding, phosphotransferase system, carbohydrate metabolism, DNA binding, DNA repair, transferase activity, oxidoreductase, purine metabolism, transcription anti-termination, transcription regulation and other related activities.We proved that the predicted protein structure quality, resolution, density and error plot values by QMEAN analysis. Based on these results, only two differentially expressed proteins under sap stress showed that the significant results, which were N-acetylgalactosamine-specific phosphotransferase enzyme IIB component 1, PTPB1_ECOLI and DinI-like protein Z3305/ECs2939 in prophage CP-933VDINI1_ECO57. In case of wine stress, the differentially expressed proteins were Transcription anti-termination protein RFAH- ECO57 NusA and PUR7-eco24- phosphoribosylamidazole-succinocarboxamide synthase showed significant results. ProtParam analysis indicating that the multiple physico-chemical characters of differentially expressed proteins were differed and compared. The phylogenetic tree represents the relationship in-between the differentially expressed proteins, were showed siblings (related) as well as monophytic clade.

Small regulatory RNAs in lambdoid bacteriophages and phage-derived plasmids: Not only antisense

Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.

A Folding-Based Approach for the Luminescent Detection of a Short RNA Hairpin

We report the rational design of a 20-mer basic peptide, derived from the transcriptional antitermination protein N of bacteriophage P22, equipped with a luminescent DOTA[Tb(3+)] macrocyclic complex and a sensitizing tryptophan antenna. Folding of this peptide into an α helical conformation, which occurs upon binding to its target boxB RNA hairpin, results in a large increase in luminescence emission. Therefore, the peptide construct works as a highly sensitive and selective probe for this RNA hairpin.

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides β-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

Structure of the RBD-PRDI fragment of the antiterminator protein GlcT

GlcT is a transcriptional antiterminator protein that is involved in regulation of glucose metabolism in Bacillus subtilis. Antiterminator proteins bind specific RNA sequences, thus preventing the formation of overlapping terminator stem-loops. The structure of a fragment (residues 3-170) comprising the RNA-binding domain (RBD) and the first regulatory domain (PRDI) of GlcT was solved at 2.0 Å resolution with one molecule in the asymmetric unit. The two domains are connected by a helical linker. Their interface is mostly constituted by hydrophobic interactions.

Cloning and biochemical analysis of β-glucoside utilization (bgl) operon without phosphotransferase system in Pectobacterium carotovorum subsp. carotovorum LY34

β-Glucosidases are widespread in bacteria and involved in the metabolism of various carbohydrate substrates. Studying of β-glucoside utilization (bgl) operons on operon of Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34) will help us understanding how β-glucoside utilization (bgl) operon can cooperate with other systems in bacterium caused soft-rot disease. Pcc LY34 causes soft-rot disease in plants and expresses multiple enzymatic forms of β-glucosidases. To fully explore the β-glucoside utilization system in Pcc LY34, we have isolated a bgl operon from a genomic library for screening of β-glucosidase activities. Sequence analysis of a 3050bp cloned DNA fragment (accession number AY870655) shows two open reading frames (bglY and bglK) that are predicted to encode proteins of 474 and 278 amino acid residues, respectively. Pair wise similarity analysis suggests BglY is a beta-glucosidase (a member of glycosyl hydrolase family 1) and BglK is a transcriptional antiterminator protein. bglY promoter region contains an inverted repeat sequence similar to transcriptional terminator. Different from other four β-glucoside utilization operons of Pcc LY34 strain, BglY contains signal peptide sequences as extracellular β-glucosidase. Comparisons of five β-glucoside utilization operons of Pcc LY34 strain showed that bglYK operon does not have phosphotransferase system domain which are responsible for sugar transportation. BglY shares 33–44% identity with other four β-glucosidases of Pcc LY34 strain. Enzyme assay showed that purified BglY enzyme hydrolyzed salicin, arbutin, pNPG, and MUG, and exhibited maximal activity at pH 7.0 and 40°C. This activity was enhanced Mg2+. Site-directed mutagenesis revealed E166 and E371 are critical of BglY's β-glucosidase activity.

Solution NMR structure of Lin0431 protein from Listeria innocua reveals high structural similarity with domain II of bacterial transcription antitermination protein NusG

Lin0431 protein from Listeria innocua (UniProtKB/TrEMBL ID {type:entrez-protein,attrs:{text:Q92EM7,term_id:81527521,term_text:Q92EM7}}Q92EM7/Q92EM7_LISIN) was selected as a target of the Northeast Structural Genomics Consortium (target ID: LkR112). Here, we present the high-quality NMR solution structure of this protein which is the first representative for a member of DUF1312 domain family. Lin0431 protein exhibits a β-sandwich topology. Four anti-parallel β-strands form one face of the sandwich and the other three anti-parallel β-strands together with a short α-helix form the other face of the sandwich. Structure alignment by Dali reveals an unexpected structural similarity with domain II of NusG from Aquifex aeolicus. Analyses of the electrostatic protein surface potential and searches for protein surface cavities reveal the conserved basic charged surface cavities of both the Lin0431 and domain II of AaeNusG, suggesting they may bind the negatively charged nucleic acids and/or and other binding partners. The high structural similarity and similar surface features, despite the lack of recognizable sequence similarity, between Lin0431 and AaeNusG domain II suggest that the domain II of NusG and DUF1312 domains have a homologous relationship and may share similar biochemical functions.

Activation of the LicT Transcriptional Antiterminator Involves a Domain Swing/Lock Mechanism Provoking Massive Structural Changes

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 A resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ' interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.

Evidence that the KH RNA-binding Domains Influence the Action of the E. coli NusA Protein

The NusA transcription elongation protein, which binds RNA, contains sequences corresponding to the S1 and KH classes of identified RNA binding domains. An essential function in E.coli, NusA is also one of the host factors required for action of the N transcription antitermination protein of λ. Tandem KH domains have been identified downstream of the S1 domain. We changed the first Gly to Asp of the GXXG motif, a tetrapeptide diagnostic of KH domains, of both NusA KH domains. The change in the first, G253D, has a large effect, while the change in the second, G319D, has a small effect on NusA action. The changes in both KH domains interfere with NusA binding to RNA. A change of a highly conserved Arg in the S1 domain, R199A, has previously been reported to interfere with RNA binding while exerting a small effect on NusA action. However, a nusA allele with both the R199A and G319D changes encodes a functionally inactive NusA protein. These studies provide direct evidence that the both KH as well as the S1 RNA binding domains are important for NusA action in support of bacterial viability as well as transcription antitermination mediated by the λ N protein.

Crystal structure of an activated form of the PTS regulation domain from the LicT transcriptional antiterminator

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It belongs to a newly characterized family of bacterial regulators whose activity is controlled by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). LicT contains an N-terminal RNA-binding domain (56 residues), and a PTS regulation domain (PRD, 221 residues) that is phosphorylated on conserved histidines in response to substrate availability. Replacement of both His207 and His269 with a negatively charged residue (aspartic acid) led to a highly active LicT variant that no longer responds to either induction or catabolite repression signals from the PTS. In contrast to wild type, the activated mutant form of the LicT regulatory domain crystallized easily and provided the first structure of a PRD, determined at 1.55 A resolution. The structure is a homodimer, each monomer containing two analogous alpha-helical domains. The phosphorylation sites are totally buried at the dimer interface and hence inaccessible to phosphorylating partners. The structure suggests important tertiary and quaternary rearrangements upon LicT activation, which could be communicated from the protein C-terminal end up to the RNA-binding domain.

The structure of the transcriptional antiterminator NusB from Escherichia coli.

We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli. The structure reveals a novel, all alpha-helical protein fold. NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions. Residues that are highly conserved among homologs stabilize the protein core. The solution structure of E. coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E. coli NusB reported earlier.

The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription.

The M2 mRNA of human respiratory syncytial virus (RSV) contains two overlapping ORFs, encoding the transcription antitermination protein (M2-1) and the 90-aa M2-2 protein of unknown function. Viable recombinant RSV was recovered in which expression of M2-2 was ablated, identifying it as an accessory factor dispensable for growth in vitro. Virus lacking M2-2 grew less efficiently than did the wild-type parent in vitro, with titers that were reduced 1, 000-fold during the initial 2-5 days and 10-fold by days 7-8. Compared with wild-type virus, the intracellular accumulation of RNA by M2-2 knockout virus was reduced 3- to 4-fold or more for genomic RNA and increased 2- to 4-fold or more for mRNA. Synthesis of the F and G glycoproteins, the major RSV neutralization and protective antigens, was increased in proportion with that of mRNA. In cells infected with wild-type RSV, mRNA accumulation increased dramatically up to approximately 12-15 hr after infection and then leveled off, whereas accumulation continued to increase in cells infected with the M2-2 knockout viruses. These findings suggest that M2-2 mediates a regulatory "switch" from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly. With regard to vaccine development, the M2-2 knockout has a highly desirable phenotype in which virus growth is attenuated while gene expression is concomitantly increased.

Role of the M2-1 transcription antitermination protein of respiratory syncytial virus in sequential transcription.

M2-1 protein of human respiratory syncytial virus (RSV) is a transcription antitermination factor that is important for the efficient synthesis of full-length mRNAs as well as for the synthesis of polycistronic readthrough mRNAs, which are characteristic of nonsegmented negative-strand RNA viruses. The contributions of these effects to RSV sequential transcription were investigated with minigenomes which contained one to five genes which were either foreign marker genes or authentic RSV genes. When evaluated on a promoter-proximal gene, the effect of M2-1 on the synthesis of full-length mRNA was much greater for a long (1,212- or 1,780-nucleotide) gene (up to a 615-fold increase) than for a short (274-nucleotide) gene (less than a 2-fold increase). This was independent of whether the gene contained non-RSV or RSV-specific sequence. Once the polymerase had terminated prematurely, it was unable to reinitiate at a downstream gene. These studies also confirmed that M2-1 enhances the synthesis of polycistronic mRNAs and that the magnitude of this effect varied greatly among different naturally occurring gene junctions. The synthesis of polycistronic mRNAs, which presumably involves antitermination at the gene-end signal, required a higher level of M2-1 than did the synthesis of the corresponding monocistronic mRNAs. M2-1 did not have a comparable antitermination effect at the junction between the leader region and the first gene. In a minigenome containing the NS1 and NS2 genes in their authentic sequence context, synthesis of full-length NS1 and NS2 mRNAs in the absence of M2-1 was remarkably high (36 and 57%, respectively, of the maximum levels observed in the presence of M2-1). In contrast, synthesis of mRNA from additional downstream genes was highly dependent on M2-1. Thus, RSV has the potential for two transcription programs: one in the absence of M2-1, in which only the NS1 and NS2 genes are transcribed, and one in the presence of M2-1, in which sequential transcription of the complete genome occurs. The dependence on M2-1 for transcription was greater for a gene in the fifth position from the promoter than for one in the third position. This indicates that under conditions where M2-1 is limiting, its concentration affects the gradient of transcription. Although M2-1 was found to have profound effects on transcription, it had no effect on replication of any minigenome tested, suggesting that it is not an active participant in RNA replication or regulation of RNA replication. Finally, since a permissive RSV infection is marked by a gradual increase in the intracellular accumulation of viral proteins including M2-1, we examined the relative abundances of various mRNAs during RSV infection for evidence of temporal regulation of transcription. None was found, implying that the availability of M2-1 during a permissive infection is sufficient at all times such that its concentration does not mediate temporal regulation of gene transcription.

NMR Structure of the Bacteriophage λ N Peptide/ boxB RNA Complex: Recognition of a GNRA Fold by an Arginine-Rich Motif

The structure of the complex formed by the arginine-rich motif of the transcriptional antitermination protein N of phage λ and boxB RNA was determined by heteronuclear magnetic resonance spectroscopy. A bent α helix in N recognizes primarily the shape and negatively charged surface of the boxB hairpin through multiple hydrophobic and ionic interactions. The GAAGA boxB loop forms a GNRA fold, previously described for tetraloops, which is essential for N binding. The fourth nucleotide of the loop extrudes from the GNRA fold to enable the E. coli elongation factor NusA to recognize the N protein/RNA complex. This structure reveals a new mode of RNA–protein recognition and shows how a small RNA element can facilitate a protein–protein interaction and thereby nucleate formation of a large ribonucleoprotein complex.

Involvement of boxA Nucleotides in the Formation of a Stable Ribonucleoprotein Complex Containing the Bacteriophage λ N Protein

The association of the transcriptional antitermination protein N of bacteriophage lambda with Escherichia coli RNA polymerase depends on nut site RNA (boxA + boxB) in the nascent transcript and the host protein, NusA. This ribonucleoprotein complex can transcribe through Rho-dependent and intrinsic termination sites located up to several hundred base pairs downstream of nut. For antitermination to occur farther downstream, this core antitermination complex must be stabilized by the host proteins NusB, NusG, and ribosomal protein S10. Here, we show that the assembly of NusB, NusG, and S10 onto the core complex involves nucleotides 2-7 of lambda boxA (CGCUCUUACACA) and is a fully cooperative process that depends on the presence of all three proteins. This assembly of NusB, NusG, and S10 also requires the carboxyl-terminal region (amino acids 73-107) of N, which interacts directly with RNA polymerase. NusB and S10 assemble in the absence of NusG when lambda boxA is altered at nucleotides 8 and 9 to create a consensus version of boxA (CGCUCUUUAACA). These experiments suggest that multiple protein-protein and protein-RNA interactions are required to convert a core antitermination complex into a complete complex.

Independent Ligand-Induced Folding of the RNA-Binding Domain and Two Functionally Distinct Antitermination Regions in the Phage λ N Protein

The transcriptional antitermination protein N of bacteriophage λ binds the boxB component of the RNA enhancer nut ( boxA + boxB) and the E. coli elongation factor NusA. Efficient antitermination by N requires an RNA-binding domain (amino acids 1–22) and two activating regions for antitermination: a newly identified NusA-binding region (amino acids 34–47) that suppresses NusA's enhancement of termination, and a carboxy-terminal region (amino acids 73–107) that interacts directly with RNA polymerase. Heteronuclear magnetic resonance experiments demonstrate that N is a disordered protein. Interaction with boxB RNA induces only the RNA-binding domain of N to adopt a folded conformation, while the activating regions of the protein remain disordered in the absence of their target proteins.

Crystallization of the RNA‐binding domain of the transcriptional antiterminator protein sacy from Bacillus subtilis

Crystallization of the RNA‐binding domain of the transcriptional antiterminator protein sacy from Bacillus subtilis

LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family

Gene licS of Bacillus subtilis encodes an excreted Beta-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two- to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in beta-glucan utilization.