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 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 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.

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.

Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

scholarly journals Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence.

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849 ◽  
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

Metabolic programming of adipose tissue in female C57Bl 6 mice offspring by maternal high fat feeding and obesity results in greater adaptability to dietary fat content and resistance to high fat diet-induced glucose intolerance compared to male mice

2021 ◽  
pp. 1-15
Keyword(s):  
Gene Expression ◽  
Dietary Fat ◽  
High Fat Diet ◽  
Female Offspring ◽  
Male Offspring ◽  
High Fat ◽  
Carnitine Palmitoyl Transferase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Maternal lifestyle influence may be a factor in the worldwide prevalence of obesity and its complications, including diabetes. Studies investigating the effect of the perinatal maternal environment have produced a range of results, sometimes diametrically opposite. The present study was designed to investigate how obesity and weight gain in pregnant mice affects energy balance, body composition and glucose homeostasis in their offspring, both at a young age on standard diet and when older and fed a high-fat diet. At six weeks of age both male and female offspring from mothers fed a high fat diet had a shorter body length than those from mothers fed standard chow. In contrast to males, female offspring also contained a higher proportion of fat and had elevated circulating leptin and adiponectin. Their gonadal fat pads were heavier and contained larger adipocytes, whereas male offspring had proportionally more smaller adipocytes. Six-week-old female, but not male, offspring had increased gonadal fat gene expression of acetyl CoA carboxylase 1, the rate-limiting step in lipid biosynthesis, and decreased gene expression of carnitine palmitoyl transferase 1, the rate-limiting step in fatty acid oxidation. Maternal high fat diet had no effect on glucose tolerance in six-week-old mice, but this was achieved with higher insulin levels in females. Contrastingly, when the offspring were fed a high fat diet for three months, female, but not male, offspring were leaner than those from mothers fed standard chow. Their gonadal fat depots were lighter and the adipocytes were smaller. Female, but not male, offspring fed high fat diet had decreased gonadal fat gene expression of acetyl CoA carboxylase 1, and increased gene expression of carnitine palmitoyl transferase 1. High fat diet-induced glucose intolerance and elevated plasma insulin concentration were improved in female, but not male, offspring. Plasma leptin and adiponectin remained higher in female offspring on high fat diet with resistin levels being lower. These results suggest that the gonadal fat of female offspring is more adaptable to different levels of dietary fat exposure, increasing storage when levels are low and increasing oxidation when levels are high. This may help female offspring be more resistant to the detrimental effects of high fat diet than male mice.

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 mRNA deadenylation by Pan2–Pan3

2014 ◽  
Vol 42 (1) ◽  
pp. 184-187 ◽  
Author(s):  
Jana Wolf ◽  
Lori A. Passmore
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Mrna Degradation ◽  
Fine Tuning ◽  
Present Review ◽  
Translational Efficiency ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Poly(A) tails are important regulators of mRNA stability and translational efficiency. Cytoplasmic removal of poly(A) tails by 3′→5′ exonucleases (deadenylation) is the rate-limiting step in mRNA degradation. Two exonuclease complexes contribute the majority of the deadenylation activity in eukaryotes: Ccr4–Not and Pan2–Pan3. These can be specifically recruited to mRNA to regulate mRNA stability or translational efficiency, thereby fine-tuning gene expression. In the present review, we discuss the activities and roles of the Pan2–Pan3 deadenylation complex.

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