Processivity In Early Stages Of Transcription By T7 Rna Polymerase? [Pdf]

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The major gene product of T7 phage, T7 RNA polymerase (T7RNAP), continues to attract researchers since a long time due to its high and specific processivity with a single subunit structure and its capability of transcribing a complete gene without additional proteins. In vitro, after binding to the promoter to form a catalytically active complex, RNA polymerases abortively cycle over the first transcribed nucleotides (initial transcribed sequence or ITS) before leaving the promoter. With the bacteriophage T7 enzyme, the extent of abortive transcription varies with the nature of the ITS and with the elongation speed of the polymerase. Abstract In order to test the proposal that most nucleotide polymerases share a common active site structure and folding topology, we have generated 22 mutations of residues within motifs A, B and C of T7 RNA polymerase (RNAP). Characterization of these T7 RNAP mutants showed the following: (i) most of the mutations resulted in moderate to drastic reductions in T7 RNAP

T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA, T7 RNA polymerase will synthesize a ‘ladder’ of poly-G RNA products. At each step, the ratio of elongation to T7 RNA polymerase (RNAP) is the prototype of a class of single subunit DNA-dependent RNAPs that includes the RNAPs of related bacteriophages such as T3 and SP6, and the mitochondrial RNAPs (McAllister & Raskin, 1993). The phage enzymes are able to perform all of the steps in the transcription cycle that are carried out by multisubunit RNAPs without the

已完结文献求助详情标题 Processivity in early stages of transcription by T7 RNA polymerase T7 RNA聚合酶转录早期的持续性相关领域引用社会化媒体偶像计算机科学 Altmetrics公司抄写（语言学） RNA聚合酶情报检索万维网核糖核酸生物基因遗传学语言学哲 Abstract Two proteolytically modified forms of T7 RNA polymerase have been characterized with respect to transcription initiation and processivity. One species, denoted 80K-20K, is singly cleaved within the region of the polypeptide

T7 RNA Polymerase: Mechanism and Engineered Variants

Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A.J., & Comparison of the T7 RNA polymerase structure with that of the homologous pol I family of DNA polymerases reveals identities in the catalytic site but also differences specific to RNA polymerase function. The structure of T7 RNA polymerase presented here differs significantly from a previously published structure.

Immediately following initiation of transcription, T7 RNA polymerase enters a phase in which dissociation of the enzyme-DNA-RNA ternary complex significantly competes with elongation, a process referred to in the Escherichia coli enzyme as abortive cycling [Carpousis, A.J., & Gralla, J.D. (1980) Biochemistry 19, 3245-3253]. Compared to RNA polymerases from other bacteriophages like T3 and SP6, T7 RNA polymerase demonstrates superior transcription rate and processivity, making it preferred in laboratory settings. Its structural features provide a stable and efficient transcription platform, and its high promoter specificity minimizes off-target effects.

DNA templates modified with C2′-methoxyls at the last two nucleotides of the 5′ termini dramatically reduced nontemplated nucleotide addition by the T7 RNA polymerase from both single- and double-stranded DNA templates. This strategy was used to The transcription of early promoters A1, A2, and A3 (in class I) located in the first 2.5% of the genome helps in pulling out the template DNA from the phage head to the host cell. Transcription by host RNA polymerase halts at the terminator (Te, a rho-independent terminator) which is present in the leftmost part (~19%) of the genome.

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days. Citations are the number of other articles citing this SP6 DNA-dependent RNA polymerase, like T7 RNA polymerase, can be used to synthesize RNA sequences from short DNA templates which contain the 18 base pair promoter region. Use of SP6 polymerase extends the range of possible 5′ sequences of RNA

Processivity of proteolytically modified forms of T7 RNA polymerase
Processivity in Early Stages of Transcription by T7 RNA Polymerase
Transient State Kinetics of Transcription Elongation by T7 RNA Polymerase

During the early stages of transcription, T7 RNA polymerase forms an unstable initiation complex that synthesizes and releases transcripts 2–8 nt in length before disengaging from the promoter and isomerizing to a stable elongation complex. In this study, we used RNA⋅protein and RNA⋅DNA Sci-Hub | Processivity in early stages of transcription by T7 RNA polymerase. Biochemistry, 27 (11), 3966–3974 | 10.1021/bi00411a012 hubto open science ↓ save The 99 kDa single-subunit RNA polymerase from bacteriophage T7 (T7 RNAP) and the multisubunit cellular RNAPs share numerous functional characteristics, in spite of their being structurally unrelated. Both families of RNAPs have initiation and elongation phases of transcription, and must translocate down the DNA as synthesis proceeds, opening the

Processivity of proteolytically modified forms of T7 RNA polymerase

1988;American Chemical Society;Volume: 27;Issue: 11Linguagem: Inglês 10.1021/bi00411a012 ISSN 1943-295X Autores Craig T. Martin, Daniel K. Muller, Joseph E. Coleman Abortive cycling by T7 RNA polymerase Abortive initiation, also known as abortive transcription, is an early process of genetic transcription in which RNA polymerase binds to a DNA promoter and enters into cycles of synthesis of short mRNA transcripts which are released before the transcription complex leaves the promoter. This process occurs in both eukaryotes and Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days. Citations are

Explore the unique traits of T7 RNA polymerase! ? Discover how its high efficiency and fidelity impact transcription, molecular biology, and biotechnology. The transcription of early promoters A1, A2, and A3 (in class I) located in the first 2.5% of the genome helps in pulling out the template DNA from the phage head to the host cell. Transcription by host RNA polymerase halts at the terminator (Te, a rho-independent terminator) which is present in the leftmost part (~19%) of the genome. The RNA polymerase from enterophage T7 (T7 RNAP) has been used as a model to understand the mechanism of transcription in general, and the transition from initiation to elongation specifically, and Crystal structures, available of multiple stages of the initiation complex and of the elongation complex, offer molecular detail of the transition.

Replicative DNA polymerases are highly processive enzymes that polymerize thousands of nucleotides without dissociating from the DNA template. The recently determined structure of the Escherichia coli bacteriophage T7 DNA polymerase suggests a unique mechanism that underlies processivity, and this mechanism may generalize to other replicative Bacteriophage T7 RNA polymerase (T7 RNAP) is known to be one of the simplest enzymes catalyzing RNA synthesis. In contrast to most RNA polymerases known, this enzyme consists of one subunit and is able to carry out transcription in the absence of additional protein factors. Owing to its molecular properties, the enzyme is widely used for synthesis of specific

Abstract RNA molecules can be conveniently synthesized in vitro by the T7 RNA polymerase (T7 RNAP). In some experiments, such as cotranscriptional biochemical analyses, continuous synthesis of RNA is not desired. Here, we propose a method for a single-pass transcription that yields a single transcript per template DNA molecule using the T7 RNAP system. We

T7 RNA polymerase (RNAP) is able to traverse a variety of discontinuities in the template (T) strand of duplex DNA, including nicks, gaps, and branched junctions in which the 3′ end of the T strand is not complementary to the non-template (NT) strand. The products represent a faithful copy of the T strand, with no insertions or deletions. On double-stranded templates having

In vitro, after binding to the promoter to form a catalytically active complex, RNA polymerases abortively cycle over the first transcribed nucleotides (initial transcribed sequence or ITS) before leaving the promoter. With the bacteriophage T7 enzyme, the extent of abortive transcription varies with the nature of the ITS and with the elongation speed of the polymerase. Abstract Bacteriophage T7 RNA polymerase is a single-subunit enzyme which has a C-terminal amino acid sequence of Phe-Ala-Phe-Ala883 (FAFA883). Closely related hydrophobic sequences are present at the C termini of seven other single-subunit RNA polymerases, including the mitochondrial RNA polymerase. Mutations at any of the four C

Two proteolytically modified forms of T7 RNA polymerase have been characterized with respect to transcription initiation and processivity. One species, denoted 80K-20K, is singly cleaved within the region of the polypeptide chain between amino acids 172 and 180. The second species, denoted 80K, is g The T7 RNA Polymerase Intercalating Hairpin is Important for Promoter Opening During Initiation but not for RNA Displacement or Transcription Bubble Stability During Elongation Biochemistry 40, 3882-3890. INTRODUCTION T7 RNA polymerase possesses all of the fundamental features of an RNA polymerase. It initiates transcription at a unique position in its promoter, it is characterized by an early less-processive abortive cycling phase, it then proceeds to a stably elongating complex and it terminates at specific sequences. As such, it serves as an ideal model system in which to

RNA is playing an ever-growing role in molecular biology and biomedicine due to the many ways it influences gene expression and its increasing use in modern therapeutics. Hence, production of RNA molecules in large quantity and high purity has become essential for advancing basic scientific research and for developing next-generation therapeutics. T7 RNA During transcription initiation, RNA polymerases appear to retain promoter interactions while transcribing short RNAs that are frequently released from the complex. Upon transition to elongation, the polymerase releases promoter and forms a stable elongation complex. Little is known about the changes in polymerase conformation or polymerase:DNA Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days. Citations are

The structures of T7 RNA polymerase (T7 RNAP) captured in the initiation and elongation phases of transcription, as well as an intermediate stage provide insights into how this RNA polymerase protein can initiate RNA synthesis and synthesize 7–10 nucleotides of RNA while remaining bound to the DNA promoter site.

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