skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Transcription yield of fully 2;-modified RNA can be increased by the addition of thermostabilizing Variants to T7 RNA polymerase

Authors:
; ; ; ; ; ; ;  [1];  [2];  [2]
  1. Texas
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1234731
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nucleic Acids Research; Journal Volume: 43; Journal Issue: (15) ; 09, 2015
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Meyers, Adam J., Garry, Daniel J., Hall, Bradley, Byrom, Michelle M., McDonald, Hannah G., Yang, Xu, Yin, Y. Whitney, Ellington, Andrew D., Texas-MED), and Altermune). Transcription yield of fully 2;-modified RNA can be increased by the addition of thermostabilizing Variants to T7 RNA polymerase. United States: N. p., 2016. Web. doi:10.1093/nar/gkv734.
Meyers, Adam J., Garry, Daniel J., Hall, Bradley, Byrom, Michelle M., McDonald, Hannah G., Yang, Xu, Yin, Y. Whitney, Ellington, Andrew D., Texas-MED), & Altermune). Transcription yield of fully 2;-modified RNA can be increased by the addition of thermostabilizing Variants to T7 RNA polymerase. United States. doi:10.1093/nar/gkv734.
Meyers, Adam J., Garry, Daniel J., Hall, Bradley, Byrom, Michelle M., McDonald, Hannah G., Yang, Xu, Yin, Y. Whitney, Ellington, Andrew D., Texas-MED), and Altermune). Tue . "Transcription yield of fully 2;-modified RNA can be increased by the addition of thermostabilizing Variants to T7 RNA polymerase". United States. doi:10.1093/nar/gkv734.
@article{osti_1234731,
title = {Transcription yield of fully 2;-modified RNA can be increased by the addition of thermostabilizing Variants to T7 RNA polymerase},
author = {Meyers, Adam J. and Garry, Daniel J. and Hall, Bradley and Byrom, Michelle M. and McDonald, Hannah G. and Yang, Xu and Yin, Y. Whitney and Ellington, Andrew D. and Texas-MED) and Altermune)},
abstractNote = {},
doi = {10.1093/nar/gkv734},
journal = {Nucleic Acids Research},
number = (15) ; 09, 2015,
volume = 43,
place = {United States},
year = {Tue Jul 05 00:00:00 EDT 2016},
month = {Tue Jul 05 00:00:00 EDT 2016}
}
  • The authors have used a self-cleaving RNA molecule (a hammerhead') to study the length-dependent folding of RNA produced during transcription by T7 RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues, 3{prime}-deoxynucleoside triphosphates. When the nascent transcription attains the minimum length required for the hammerhead' domain of the transcript to fully emerge from the ternary complex, the hammerhead' structure forms and self-cleaves, producing a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible: the sequencing ladder is compared to that generated by using a noncleavingmore » control template. They have shown that 13 nucleotides past the cleavage point must be synthesized before the transcript can self-cleave in the ternary complex whereas RNA freed from the complex by heating can cleave with only 3 or more nucleotides present beyond the cleavage site. The results indicate that RNA in T7 RNA polymerase is not free of steric interactions in the ternary complex and not available for structure formation until it is at least 10 bases away from the site of polymerization. The results suggest that the maximum possible length of the RNA-DNA hybrid in the ternary complexes is 10. The relevance of the results in comparisons with other RNA polymerases, especially Escherichia coli RNA polymerase, is discussed.« less
  • The authors previously described the purification and characterization of E{sub 1}BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E{sub 1}BF was selectively phosphorylated. The labeled phosphate could be removed from the E{sub 1}BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E{sub 1}BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluentmore » with ({gamma}-{sup 32}P)ATP resulted in the selective labeling o the 79-kDa band. The E{sub 1}BF-associated protein kinase did not phosphorylate casein or histone H1. These data demonstrate that (1) polymerase I promoter-binding factor E{sub 1}BF contains an intrinsic substrate-specific protein kinase and (2) E{sub 1}BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.« less
  • The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and refined at 2.2-{angstrom} resolution. The protein folds into an {alpha}/{beta}-sheet structure that has a prominent cleft. A zinc atom is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Alignment of the zinc ligands ofmore » T7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amidase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the cleft and a surface that appears to be the site of binding to T7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.« less