Postinitiation transcriptional control in Drosophila melanogaster

1990 ◽  
Vol 10 (11) ◽  
pp. 6041-6045
Author(s):  
A E Rougvie ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Transcriptional Regulation ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Drosophila hsp70 genes have an RNA polymerase II molecule paused at their 5' ends in uninduced cells. In this study we have shown that this pausing also occurs on other heat shock and constitutively expressed genes. We propose that a rate-limiting step in early elongation occurs in many Drosophila genes and may be a target for transcriptional regulation.

scholarly journals Postinitiation transcriptional control in Drosophila melanogaster.

1990 ◽  
Vol 10 (11) ◽  
pp. 6041-6045 ◽  
Author(s):  
A E Rougvie ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Transcriptional Regulation ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Drosophila hsp70 genes have an RNA polymerase II molecule paused at their 5' ends in uninduced cells. In this study we have shown that this pausing also occurs on other heat shock and constitutively expressed genes. We propose that a rate-limiting step in early elongation occurs in many Drosophila genes and may be a target for transcriptional regulation.

RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene

1991 ◽  
Vol 11 (10) ◽  
pp. 5285-5290
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Intermediate Level ◽  
Hsp70 Gene ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Initiation Of Transcription ◽  
Rate Limiting

An RNA polymerase II molecule is associated with the 5' end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides (A. E. Rougvie and J. T. Lis, Cell 54:795-804, 1988). Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here we report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5' end of hsp70 in cells exposed to the intermediate heat shock temperatures of 27 and 30 degrees C. At 30 degrees C, each copy of hsp70 was transcribed approximately five times during the 25-min heat shock that we used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

scholarly journals RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene.

1991 ◽  
Vol 11 (10) ◽  
pp. 5285-5290 ◽  
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Intermediate Level ◽  
Hsp70 Gene ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Initiation Of Transcription ◽  
Rate Limiting

An RNA polymerase II molecule is associated with the 5' end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides (A. E. Rougvie and J. T. Lis, Cell 54:795-804, 1988). Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here we report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5' end of hsp70 in cells exposed to the intermediate heat shock temperatures of 27 and 30 degrees C. At 30 degrees C, each copy of hsp70 was transcribed approximately five times during the 25-min heat shock that we used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

scholarly journals A Novel Initiation Pathway in Escherichia Coli Transcription

2016 ◽  
Author(s):  
Eitan Lerner ◽  
SangYoon Chung ◽  
Benjamin L. Allen ◽  
Shuang Wang ◽  
Jookyung J. Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Magnetic Tweezer ◽  
Delayed Initiation ◽  
Rate Limiting ◽  
Kinetics Of

AbstractInitiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes.Significance:Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster

1985 ◽  
Vol 5 (8) ◽  
pp. 2009-2018
Author(s):  
D S Gilmour ◽  
J T Lis
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Characterization ◽  
Heat Shock Gene ◽  
Transcriptional Level ◽  
Intact Cells

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

scholarly journals Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment.

1997 ◽  
Vol 17 (12) ◽  
pp. 6898-6905 ◽  
Author(s):  
H Xiao ◽  
J T Lis ◽  
K T Jeang
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Transcriptional Unit ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Adenovirus E1b ◽  
Rnap Ii ◽  
Rate Limiting ◽  
Hiv 1

Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.

scholarly journals A yeast TATA-binding protein mutant that selectively enhances gene expression from weak RNA polymerase II promoters.

1997 ◽  
Vol 17 (5) ◽  
pp. 2888-2896 ◽  
Author(s):  
W S Blair ◽  
B R Cullen
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Mrna Transcription ◽  
Highly Active ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

We describe a unique gain-of-function mutant of the TATA-binding protein (TBP) subunit of Saccharomyces cerevisiae TFIID that, at least in part, renders transcriptional transactivators dispensable for efficient mRNA expression. The yTBPN69S mutant enhances transcription from weaker yeast promoter elements by up to 50-fold yet does not significantly increase gene expression directed by highly active promoters. Therefore, this TBP mutant and transcriptional transactivators appear to affect a common rate-limiting step in transcription initiation. Consistent with the hypothesis that this step is TFIID recruitment, tethering of TBP to a target promoter via a heterologous DNA binding domain, which is known to bypass the need for transcriptional transactivators, also nullifies the enhancing effect exerted by the N69S mutation. These data provide genetic support for the hypothesis that TFIID recruitment represents a rate-limiting step in the initiation of mRNA transcription that is specifically enhanced by transcriptional transactivators.

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