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.

scholarly journals Nuclear Import of the TATA-binding Protein: Mediation by the Karyopherin Kap114p and a Possible Mechanism for Intranuclear Targeting

1999 ◽  
Vol 145 (7) ◽  
pp. 1407-1417 ◽  
Author(s):  
Lucy F. Pemberton ◽  
Jonathan S. Rosenblum ◽  
Günter Blobel
Keyword(s):  
Family Member ◽  
Recombinant Proteins ◽  
Nuclear Import ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Transcriptional Complexes ◽  
Rate Limiting

Binding of the TATA-binding protein (TBP) to the promoter is the first and rate limiting step in the formation of transcriptional complexes. We show here that nuclear import of TBP is mediated by a new karyopherin (Kap) (importin) family member, Kap114p. Kap114p is localized to the cytoplasm and nucleus. A complex of Kap114p and TBP was detected in the cytosol and could be reconstituted using recombinant proteins, suggesting that the interaction was direct. Deletion of the KAP114 gene led to specific mislocalization of TBP to the cytoplasm. We also describe two other potential minor import pathways for TBP. Consistent with other Kaps, the dissociation of TBP from Kap114p is dependent on RanGTP. However, we could show that double stranded, TATA-containing DNA stimulates this RanGTP-mediated dissociation of TBP, and is necessary at lower RanGTP concentrations. This suggests a mechanism where, once in the nucleus, TBP is preferentially released from Kap114p at the promoter of genes to be transcribed. In this fashion Kap114p may play a role in the intranuclear targeting of TBP.

scholarly journals Analysis of Efficiency and Fidelity of HIV-1 (+)-Strand DNA Synthesis Reveals a Novel Rate-limiting Step during Retroviral Reverse Transcription

2000 ◽  
Vol 276 (9) ◽  
pp. 6711-6719 ◽  
Author(s):  
Matthias Götte ◽  
Masanori Kameoka ◽  
Nathan McLellan ◽  
Luciano Cellai ◽  
Mark A. Wainberg
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Hiv 1

Recruitment of the TATA-binding protein to the HIV-1 promoter is a limiting step for Tat transactivation

AIDS ◽  
1998 ◽  
Vol 12 (15) ◽  
pp. 1957-1964 ◽  
Author(s):  
Barbara Majello ◽  
Giuliana Napolitano ◽  
Luigi Lania
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
Limiting Step ◽  
Hiv 1

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.

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 TATA-Binding Protein-Interacting Protein 120, TIP120, Stimulates Three Classes of Eukaryotic Transcription via a Unique Mechanism

1999 ◽  
Vol 19 (12) ◽  
pp. 7951-7960 ◽  
Author(s):  
Yasutaka Makino ◽  
Shingo Yogosawa ◽  
Kentaro Kayukawa ◽  
Frederic Coin ◽  
Jean-Marc Egly ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Kinetic Studies ◽  
Tata Binding Protein ◽  
Binding Activity ◽  
Mobility Shift ◽  
Basal Transcription ◽  
Interacting Protein ◽  
Rnap Ii

ABSTRACT We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.

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.

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