A Protein Factor Influencing Mammalian RNA Polymerase B

1973 ◽  
pp. 163-177 ◽  
Author(s):  
Hans Stein ◽  
Horst Hameister ◽  
Claude Kedinger
Keyword(s):  
Rna Polymerase ◽  
Protein Factor

scholarly journals Chemical transcription roadblocking for nascent RNA display

2019 ◽  
Author(s):  
Eric J. Strobel ◽  
John T. Lis ◽  
Julius B. Lucks
Keyword(s):  
Rna Polymerase ◽  
Rna Structure ◽  
Triethylene Glycol ◽  
Rna Molecules ◽  
Protein Factor ◽  
Nascent Rna ◽  
Dna Strand ◽  
And Function ◽  
Dna Template

AbstractSite-specific arrest of RNA polymerase is fundamental to several technologies that measure RNA structure and function. Current in vitro transcription ‘roadblocking’ approaches inhibit transcription elongation using a protein blockade bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires the inclusion of a protein factor that is extrinsic to the minimal in vitro transcription reaction. In this work, we show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification efficiently and stably halts Escherichia coli RNA polymerase transcription. To facilitate diverse applications of chemical transcription roadblocking, we establish a simple and sequence-independent method for the preparation of internally modified double-stranded DNA templates by sequential PCR and translesion synthesis. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables nascent RNA molecules to be displayed from RNA polymerase in a minimal in vitro transcription reaction.

scholarly journals Repeat-specific functions for the C-terminal domain of RNA polymerase II in budding yeast

2018 ◽  
Author(s):  
Michael Babokhov ◽  
Mohammad M. Mosaheb ◽  
Richard W. Baker ◽  
Stephen M. Fuchs
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Budding Yeast ◽  
Distal Region ◽  
Terminal Domain ◽  
Protein Factor ◽  
Functional Regions ◽  
Heptad Repeats ◽  
In The Beginning

AbstractThe C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is required to regulate transcription and to integrate it with other essential cellular processes. In the budding yeast Saccharomyces cerevisiae, the CTD of Rpb1p consists of 26 conserved heptad repeats that are post-translationally modified to orchestrate protein factor binding at different stages of the transcription cycle. A long-standing question in the study of the CTD is if there are any functional differences between the 26 repeats. In this study, we present evidence that repeats of identical sequence have different functions based on their position within the CTD. We assembled plasmids expressing Rpb1p with serine to alanine substitutions in three defined regions of the CTD and measured a range of phenotypes for yeast expressing these constructs. Mutations in the beginning and middle regions of the CTD had drastic, and region-specific effects, while mutating the distal region had no observable phenotype. Further mutational analysis determined that Ser5 within the first region of repeats was solely responsible for the observed growth differences and sequencing fast-growing suppressors allowed us to further define the functional regions of the CTD. This mutational analysis is consistent with current structural models for how the RNAPII holoenzyme and the CTD specifically would reside in complex with Mediator and establishes a foundation for studying regioselective binding along the repetitive RNAPII CTD.

Recruitment of RNA polymerase to Class II CRP-dependent promoters is improved by a second upstream-bound CRP molecule

2006 ◽  
Vol 34 (6) ◽  
pp. 1075-1078 ◽  
Author(s):  
N.S. Miroslavova ◽  
J.E. Mitchell ◽  
J. Tebbutt ◽  
S.J.W. Busby
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Class Ii ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Protein Factor ◽  
Camp Receptor

Genetics and biochemistry have been exploited to investigate transcription activation by the Escherichia coli CRP (cAMP receptor protein) factor at promoters with a DNA site for CRP near position −41 and the effects of a second upstream-bound CRP molecule. We show that the upstream-bound CRP contributes to transcription activation by improving the recruitment of RNA polymerase.

RNA polymerase: Preparation and structural analysis of helical polymers

1984 ◽  
Vol 42 ◽  
pp. 152-153
Author(s):  
E. Loren Buhle ◽  
Pamela Rew ◽  
Ueli Aebi
Keyword(s):  
Structural Analysis ◽  
Rna Polymerase ◽  
Molecular Level ◽  
X Ray Diffraction ◽  
Helical Polymers ◽  
X Ray ◽  
E Coli ◽  
The Past ◽  
Ordered Arrays ◽  
Low Ionic Strength

While DNA-dependent RNA polymerase represents one of the key enzymes involved in transcription and ultimately in gene expression in procaryotic and eucaryotic cells, little progress has been made towards elucidation of its 3-D structure at the molecular level over the past few years. This is mainly because to date no 3-D crystals suitable for X-ray diffraction analysis have been obtained with this rather large (MW ~500 kd) multi-subunit (α2ββ'ζ). As an alternative, we have been trying to form ordered arrays of RNA polymerase from E. coli suitable for structural analysis in the electron microscope combined with image processing. Here we report about helical polymers induced from holoenzyme (α2ββ'ζ) at low ionic strength with 5-7 mM MnCl2 (see Fig. 1a). The presence of the ζ-subunit (MW 86 kd) is required to form these polymers, since the core enzyme (α2ββ') does fail to assemble into such structures under these conditions.

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

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