A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.

/pdf/oligoribonucleotide-synthesis-using-t7-rna-polymerase-and-q3o6uiumun.pdf

Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates

Structures of a 10-subunit yeast RNA polymerase II have been derived from two crystal forms at 2.8 and 3.1 angstrom resolution. Comparison of the structures reveals a division of the polymerase into four mobile modules, including a clamp, shown previously to swing over the active center. In the 2.8 angstrom structure, the clamp is in an open state, allowing entry of straight promoter DNA for the initiation of transcription. Three loops extending from the clamp may play roles in RNA unwinding and DNA rewinding during transcription. A 2.8 angstrom difference Fourier map reveals two metal ions at the active site, one persistently bound and the other possibly exchangeable during RNA synthesis. The results also provide evidence for RNA exit in the vicinity of the carboxyl-terminal repeat domain, coupling synthesis to RNA processing by enzymes bound to this domain.

/pdf/structural-basis-of-transcription-rna-polymerase-ii-at-2-8-51klsi7yp1.pdf

Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.

We have compiled and analyzed 263 promoters with known transcriptional start points for E. coli genes. Promoter elements (-35 hexamer, -10 hexamer, and spacing between these regions) were aligned by a program which selects the arrangement consistent with the start point and statistically most homologous to a reference list of promoters. The initial reference list was that of Hawley and McClure (Nucl. Acids Res. 11, 2237-2255, 1983). Alignment of the complete list was used for reference until successive analyses did not alter the structure of the list. In the final compilation, all bases in the -35 (TTGACA) and -10 (TATAAT) hexamers were highly conserved, 92% of promoters had inter-region spacing of 17 +/- 1 bp, and 75% of the uniquely defined start points initiated 7 +/- 1 bases downstream of the -10 region. The consensus sequence of promoters with inter-region spacing of 16, 17 or 18 bp did not differ. This compilation and analysis should be useful for studies of promoter structure and function and for programs which identify potential promoter sequences.

/pdf/analysis-of-e-coli-promoter-sequences-115zp5iur0.pdf

Analysis of E. coli promoter sequences.

https://www.cell.com/cell/pdf/0092-8674(86)90070-X.pdf

Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of Yeast

https://www.cell.com/cell/pdf/S0092-8674(00)81515-9.pdf

Crystal Structure of Thermus aquaticus Core RNA Polymerase at 3.3 Å Resolution

CONTENTS Perspectives and Summary .. Components of the System .. Location of the Promoter by RNA Polymerase .. The Closed Promoter-Polymerase Interaction .. The Closed-to-Open Promoter Transition and Analysis of the Open

Morgan Wd

Papers

Protein-Nucleic Acid Interactions in Transcription: A Molecular Analysis