The 5- flanking sequences of Drosophila tRNAArg genes control their in vitro transcription in a Drosophila cell extract.

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

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In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1989 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1989-1994 ◽

Cited By ~ 15

Author(s):

R Sen ◽

D Baltimore

Keyword(s):

B Cell ◽

Dna Sequences ◽

In Vitro Transcription ◽

Cell Extract ◽

Upstream Sequence ◽

Immunoglobulin Genes ◽

Specific Expression ◽

Sequence Element ◽

Transcription System

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

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Role of the C-terminal domains of rice (Oryza sativa L.) bZIP proteins RF2a and RF2b in regulating transcription

Biochemical Journal ◽

10.1042/bj20061375 ◽

2007 ◽

Vol 405 (2) ◽

pp. 243-249 ◽

Cited By ~ 6

Author(s):

Yi Liu ◽

Shunhong Dai ◽

Roger N. Beachy

Keyword(s):

Oryza Sativa ◽

Leucine Zipper ◽

Oryza Sativa L ◽

Activation Function ◽

In Vitro Transcription ◽

Cell Extract ◽

Rice Tungro Bacilliform Virus ◽

Basic Leucine Zipper ◽

Terminal Domains

Rice (Oryza sativa L.) transcription factors RF2a and RF2b are bZIP (basic leucine zipper) proteins that interact with, and activate transcription from the RTBV (rice tungro bacilliform virus) promoter. Here we characterize the C-terminal domains of RF2a and RF2b: these domains are rich in glutamine and proline/glutamine, respectively. Affinity pull-down assays demonstrated that the C-terminal domains of RF2a and RF2b can associate to form either homodimers or heterodimers; however, they do not interact with other domains of RF2a or RF2b. Results of in vitro transcription assays using a rice whole-cell extract demonstrate that the C-terminal domains of both RF2a and RF2b activate transcription from the RTBV promoter. In addition, dimerization of the RF2a C-terminal domain is involved in regulating the transcription activation function of RF2a. The predicted helical region within the RF2a C-terminal glutamine-rich domain was determined to be involved in inter-molecular dimerization, and contributed to the regulatory functions of RF2a in these assays.

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In Vitro Transcription: Whole-Cell Extract

Recombinant DNA Methodology ◽

10.1016/b978-0-12-765560-4.50042-3 ◽

1989 ◽

pp. 653-666

Author(s):

JAMES L. MANLEY ◽

ANDREW FIRE ◽

MARK SAMUELS ◽

PHILLIP A. SHARP

Keyword(s):

In Vitro Transcription ◽

Cell Extract ◽

Whole Cell ◽

Whole Cell Extract

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In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1989-1994.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1989-1994

Author(s):

R Sen ◽

D Baltimore

Keyword(s):

B Cell ◽

Dna Sequences ◽

In Vitro Transcription ◽

Cell Extract ◽

Upstream Sequence ◽

Immunoglobulin Genes ◽

Specific Expression ◽

Sequence Element ◽

Transcription System

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

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5′-flanking sequences that inhibit in vitro transcription of a Xenopus laevis tRNA gene

Cell ◽

10.1016/0092-8674(83)90545-7 ◽

1983 ◽

Vol 34 (3) ◽

pp. 881-890 ◽

Cited By ~ 41

Author(s):

Robert A. Hipskind ◽

Stuart G. Clarkson

Keyword(s):

Xenopus Laevis ◽

Trna Gene ◽

In Vitro Transcription ◽

Flanking Sequences

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Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4555 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4555-4560 ◽

Cited By ~ 60

Author(s):

M Woontner ◽

P A Wade ◽

J Bonner ◽

J A Jaehning

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

In Vitro Transcription ◽

Cell Extract ◽

Whole Cell ◽

Transcription System ◽

Upstream Activation Sequence ◽

Activation Sequence

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

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A discrete region centered 22 base pairs upstream of the initiation site modulates transcription of Drosophila tRNAAsn genes

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4441-4449.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4441-4449

Author(s):

A K Lofquist ◽

A D Garcia ◽

S J Sharp

Keyword(s):

Transcription Initiation ◽

In Vitro Transcription ◽

Initiation Site ◽

Trna Genes ◽

Selection Mechanism ◽

Base Pairs ◽

Discrete Region ◽

Flanking Sequences ◽

Actual Sequence

We have studied the mechanism by which 5'-flanking sequences modulate the in vitro transcription of eucaryotic tRNA genes. Using deletion and linker substitution mutagenesis, we have found that the 5'-flanking sequences responsible for the different in vitro transcription levels of three Drosophila tRNA5Asn genes are contained within a discrete region centered 22 nucleotides upstream from the transcription initiation site. In conjunction with the A-box intragenic control region, this upstream transcription-modulatory region functions in the selection mechanism for the site of transcription initiation. Since the transcription-modulatory region directs the position of the start site and the actual sequence of the transcription-modulatory region determines the level of tRNAAsn gene transcription, the possibility is raised that the transcription-modulatory region directs a transcription initiation event similar to open complex formation at procaryotic promoters.

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