Rho-dependent Transcription Termination: More Questions than Answers

Ranjan Sen,Jisha Chalissery,Sharmistha Banerjee,Irfan Bandey 한국미생물학회 2006 The journal of microbiology Vol.44 No.1

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain obscure. In the present review we have attempted to summarize ‘the knowns’ and ‘the unknowns’ of the Rho protein revealed by the recent developments in this field. An attempt has also been made to understand the physiology of Rho in the light of its phylogeny.

Expression and Characterization of RNA-dependent RNA Polymerase of Dendrolimus punctatus Tetravirus

Zhou, Liang,Zhang, Jiamin,Wang, Xiaochun,Jiang, Hong,Yi, Fuming,Hu, Yuanyang 생화학분자생물학회 2006 Journal of biochemistry and molecular biology Vol.39 No.5

Dendrolimus punctatus tetravirus (DpTV) has been identified as a new member of the genus Omegatetravirus of the family Tetraviridae that may be related serologically to Nudaurelia capensis virus ($N{\omega}V$). To establish the function of DpTV RNA genome and to better understand the mechanism of viral replication, the putative RNA-dependent RNA polymerase (RdRp) domain has been cloned and expressed in Escherichia coli. The recombinant protein was purified on a Ni-chelating HisTrap affinity column and demonstrated to initiate viral RNA synthesis in a primer-independent manner but not by terminal nucleotidyle transferase activity in the presence of $Mg^{2+}$ and RNA template. Mutation of the GDD to GAA interferes with the residues at the polymerase active site and metal ions, and thus renders the polymerase inactive.

Expression and characterization of RNA-dependent RNA polymerase of Ectropis obliqua virus

( Mei Juan Lin ),( Shan Ye ),( Yi Xiong ),( Da Wei Cai ),( Jia Min Zhang ),( Yuan Yang Hu ) 생화학분자생물학회 2010 BMB Reports Vol.43 No.4

Replication of positive-strand RNA virus is mediated by a virus- encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Ectropis obliqua virus (EoV), a newly identified insect virus belonging to the family Iflaviradae, we expressed the RNA polymerase domain in Escherichia coli and purified it on a Ni-chelating HisTrap affinity column. It is demonstrated that EoV RdRp initiated RNA synthesis in a primer- and poly (A)-dependent manner in vitro. Furthermore, the effect of primer concentration, temperature, metal ions (Mg2+, Mn2+, and K+) on enzymatic activity were determined. Our study represented a first step towards understanding the mechanism of EoV replication. [BMB reports 2010; 43(4): 284-290]

Transformation efficiency and transgene expression level in markerfree RDR6-knockdown transgenic tobacco plants

Tatsuya Mikami,Yuta Saeki,Sayaka Hirai,Mayuko Shimokawa,Yukiko Umeyama,Yusaku Kuroda,Hiroaki Kodama 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.6

RNA silencing is a sequence-specific form of epigenetic regulation that targets invasive nucleic acids. RNA-dependent RNA polymerase6 (RDR6) converts target RNA molecules, such as transgene transcripts, into double-stranded RNAs (dsRNAs) during posttranscriptional gene silencing (PTGS). Then, these dsRNAs are processed into small RNAs that guide sequencespecific RNA degradation. T-DNA-derived small RNAs are generated during the transfer of T-DNA from Agrobacterium to plant cells and compromise the function of the genes in the T-DNA. In the present study, we produced selection-markerfree transgenic tobacco plants using the MAT vector system, and expression of the tobacco RDR6 gene (NtRDR6) was suppressed using inverted-repeat-induced PTGS. Reduced expression of the NtRDR6 gene improved the transient expression of the transgene in the agroinfiltrated leaves and enhanced the production of hairy roots after infection with Agrobacterium containing a root-inducing T-DNA. The expression level of the sense transgene was determined in individual hairy roots, and knockdown of the NtRDR6 gene did not affect the distribution of the expression levels in individual transformants. These results indicate that NtRDR6 partially inhibited T-DNA function during T-DNA transfer but did not affect the expression of the transgene in stable transformants, except in transformants showing sense-transgene-induced PTGS.

Rho-dependent Transcription Termination: More Questions than Answers

Banerjee Sharmistha,Chalissery Jisha,Bandey Irfan,Sen Ranjan The Microbiological Society of Korea 2006 The journal of microbiology Vol.44 No.1

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain obscure. In the present review we have attempted to summarize 'the knowns' and 'the unknowns' of the Rho protein revealed by the recent developments in this field. An attempt has also been made to understand the physiology of Rho in the light of its phylogeny.

Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase in complex with 2-thiouridine or ribavirin

Alam, I.,Lee, J.H.,Cho, K.J.,Han, K.R.,Yang, J.M.,Chung, M.S.,Kim, K.H. Academic Press 2012 Virology Vol.426 No.2

Murine norovirus-1 (MNV-1) shares many features with human norovirus (HuNoV) and both are classified within the norovirus genus of Caliciviridae family. MNV-1 is used as the surrogate for HuNoV research since it is the only form that can be grown in cell culture. HuNoV and MNV-1 RNA dependent RNA polymerase (RdRp) proteins with the sequence identity of 59% show essentially identical conformations. Here we report the first structural evidence of 2-thiouridine (2TU) or ribavirin binding to MNV-1 RdRp, based on the crystal structures determined at 2.2A and 2.5A resolutions, respectively. Cellular and biochemical studies revealed stronger inhibitory effect of 2TU on the replication of MNV-1 in RAW 264.7 cells, compared to that of ribavirin. Our complex structures highlight the key interactions involved in recognition of the nucleoside analogs which block the active site of the viral RNA polymerase.

Expression of RNA dependent RNA polymerase gene from BQCV in honey bee for application of monoclonal antibody generation

Giang Huong Thi Luong,Byoungsu Yoon 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Black queen cell virus (BQCV), one of the most prevalent viruse, causes the death of queen larvae and pupae. The RNA-dependent RNA polymerases (RdRPs) are central components in the life cycle of RNA viruses that catalyzes the replication of RNA from an RNA template without DNA stage. Inhibition of RdRP gene is importantly significant for application of monoclonal antibody generation as a diagnosis tool for identifying BQCV infection in honey bee..In this study, the presence of BQCV in honey bee samples was confirmed by PCR using BQCV F/R primer set to multiply of 700 bp DNA fragment. For ampification of BQCV Rdrp gene, a primer set attached BamHI/SalI restriction site was designed based on the best homogenization between BQCV RdRP sequences in NCBI, a PCR product containing BQCV RdRP gene with 1576 bp in length was amplified. Furthermore, BQCV RdRP gene will be cloned into pBlueXcm vector for future researches.

한국에서 분리한 사람 Caliviviruses의 RnA-dependent RNA Polymerase 유전자의 다양성

최종환,한동표,이현우,손준,김현원 연세대학교 원주의과대학 1997 원주의대논문집 Vol.10 No.1

한국에 산재하는 사람 Caliciviruses의 다양한 유전자군 : 1987 - 1994년

남기범,김지애,양재명,김경희 대한바이러스학회 1997 Journal of Bacteriology and Virology Vol.27 No.2

Sequence comparison of the RNA-dependent RNA polymerase of human caliciviruses (HuCVs) from Korean children with gastroenteritis revealed significant genetic variation among them. cDNA clones were produced from the HuCVs collected from pediatric population during a period of 1987-1994. The application of reverse transcription-polymerase chain reaction (RT- PCR) using primers directed to the RNA-dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 13.7% of HuCVs yielded PCR products of similar size to the NV prototype, NV8Flla/68/US, with exceptions of HuV185/87/Korea and HuCV1115/90/Korea. Computer analyses showed that the PCR products had a continuous protein encoding frame on the positive strand, and contained GLPSG and YGDD amino acid motifs at the predicted distance from primers. Alignment of the amino acid sequences of HuCVs with previously published sequences for Snow Mountain agent (SMA), NV, and Sapporo/82/Japan indicated that these strains can be divided into four major genogroups. There were 10 (45%) SMA-like CVs, one (4.5%) NV-like HuCVs, two (9%) Sapporo-like HuCVs, and nine (41%) unidentified HuCVs. This fourth genogroup should be investigated further. HuCV185/87/Korea and HuCV 1115/90/Korea, Sapporo-like CVs, were genetically distinct from previously characterized HuCVs and more closely related to known animal CVs. One of the animal CV-like strain, HuCV 185/87/Korea, showed nucleotide and amino acid homology of only 67% and 73% with the prototype Sapporo/82/Japan. Further characterization of animal and human CV genomes and studies of possible cross-transmission of CVs from animals to humans are likely to be beneficial in understanding the epidemiology of HuCVs.

Nucleotide Sequence Analysis of the RNA-dependent RNA Poymerase Gene of Infectious Pancreatic Necrosis Virus DRT Strain

CHUNG, HYE KYUNG,LEE, SEONG HUN,KIM, SOO YOUNG,LEE, HYUNG HOAN 한국미생물 · 생명공학회 1994 Journal of microbiology and biotechnology Vol.4 No.4

To determine the nucleotide sequence of the ds RNA segment B containing the RNA dependent RNA polymerase (RdRp) gene of the DRT strain of infectious pancreatic necrosis virus (IPNV), the cDNA of the ds RNA segment B of the DRT strain of IPNV was synthesized using the reverse transcriptase (RT)-polymerase chain reaction (PCR) and its cDNA nucleotide sequence was determined. The DRT segment B was 2,783 by long and contained only a single long open reading frame (ORF) of 2,535 by in length. This ORF nucleotides encoded the VP1 protein, the putative RdRp of IPNV. The VP1 protein consisted of 845 amino acids. The molecular weight of the RdRp, as deduced from the nucleotide sequence, is 94,426. The nucleotide sequence of the ORF of the DRT showed 89.7% homology to the jasper strain, but 80.8% to the Sp strain. The amino acid sequence of the ORF of the DRT showed 97.6% homology to the jasper strain, but 88.7% to the Sp strain. The conserved GTP-binding motif was detected in VP1 protein.