Topography of transcription: path of the leading end of nascent RNA through the Escherichia coli transcription complex.

Abstract

A cleavable dinucleotide photoaffinity reagent was prepared and used to map the path of the leading end of the RNA transcript across the surface of Escherichia coli RNA polymerase/ T7 DNA transcription complexes. By using 5'-(4-azidophenacylthio)phosphoryladenylyl(3'-5')uridine, transcription was specifically initiated at the Al promoter of bacteriophage T7 DIll or D123 DNA. Transcription complexes containing radiolabeled RNA chains of various lengths (4-116 nucleotides) were prepared, and the 5' end of the RNA transcript was then covalently attached to the nearby polymerase subunits or DNA by irradiation with UV light. The photoaffinity-labeled enzyme subunits and DNA were separated, the radiolabeled RNAs were cleaved from each, and the lengths and sequences of RNA attached to each component were determined. The leading end of RNA chains up to 12 bases long was found to label the DNA and the ,3 and /3' subunits of RNA polymerase, with more than 90% of the label going to the DNA. When the RNA transcript reached 12 bases in length, the 5' end diverged from the DNA and only the /8 and /3' enzyme subunits were labeled thereafter. These two subunits were heavily labeled by RNA chains 12 to as many as 94 bases long. No signifitant labeling of the a subunit occurred. The a subunit was not labeled by RNAs longer than the trinucleotide.DNA-dependent RNA polymerases play a key role in gene expression. Escherichia coli RNA polymerase, the most extensively studied, contains five major subunits and has a Mr of approximately 454,000 (1). The "core" enzyme consists of subunits A3' (Mr, 160,000), /3 (Mr, 151,000), and two a subunits (Mr,36,000) and is capable of RNA elongation after transcription has been initiated. The holoenzyme contains the core plus the dissociable subunit or (Mr, 70,000) have been determined. Our method involves the use of a cleavable photoaffinity probe, containing both a photosensitive azide moiety for covalent attachment and a selectively cleavable S-P bond for subsequent removal of the attached oligonucleotide. We chose to prepare the dinucleotide photoaffinity reagent we used (Fig. 1) for three reasons: dinucleotides initiate transcription much more efficiently than mononucleotides (26); the aromatic azide moiety produces a chemically reactive, relatively nonselective nitrene (27) upon illumination with wavelengths greater than 300 nm; and the S-P bond was found to be stable under various conditions, although it could be cleanly hydrolyzed in the presence of organomercurials.At the initiation of transcription, the photoaffinity probe is incorporated into the leading (5') end of a nascent RNA chain. Because addition of nucleoside triphosphates occurs at the 3' end of the growing RNA chain in the elongation site of the enzyme, the 5' end must move out over the surface of the enzyme-DNA complex as transcription proceeds. Transcription complexes containing RNA chains of almost every length from 4 to 116 nucleotides have been prepared by using RNA chain terminators that substitute for ATP, CTP...

Termination-altering amino acid substitutions in the beta' subunit of Escherichia coli RNA polymerase identify regions involved in RNA chain elongation.

“…However, there are good reasons to suspect that [3' also plays a role in transcript elongation and its regulation. Several studies have documented cross-linking between [3' and both the DNA template (Okada et al 1978;Chenchick et al 1982) and the nascent RNA transcript (Hanna and Meares 1983;Dissinger and Hanna 1990;Borukhov et al 1991). Substitutions in the Pol II homolog of [3' confer resistance to a-amanitin (Area), a fungal toxin that blocks transcript elongation (Sentenac 1985).…”

In Vitro Analysis of Elongation and Termination by Mutant RNA Polymerases with Altered Termination Behavior

“…The binding pocket for the nucleoside triphosphate (NTP) substrate spans both subunits (18,19,40,44,55). Both subunits also seem to make up the protein surfaces that contact the DNA template (5,16) and the nascent RNA product (6,14,20,32,45). Binding interactions between these two subunits and the DNA and RNA components of the transcription complex most likely contribute to the extreme stability and remarkable processivity of the ternary complex.…”

Identification of a nucleic acid‐binding region within the largest subunit ofDrosophila melanogasterRNA polymerase II

“…The two large subunits of eukaryotic RNA polymerases are the structural and functional homologues of the P' and P subunit of the E. coli 223 RNA polymerase and form the major part of the catalytic site. As shown by photochemical cross-linking experiments and antibody studies, both large subunits are involved in the interaction with the DNA template and the nascent RNA chain (Hillel & Wu, 1978;Park et al, 1980;Carroll & Stollar, 1982, 1983Huet et al, 1982;Gundelfinger, 1983;Hanna & Meares, 1983;Bartholomew et al, 1986). Nonspecific DNA binding of the largest subunits of Drosophila hydei RNA polymerases I, 11, and I11 and of RNA polymerases I1 of Ehrlich ascites tumor cells and of chicken myeloblastosis cells has also been demonstrated by Southwestern blotting, a technique that has been successfully used for the analysis of nonspecific as well as specific DNA binding by a large number of proteins (Bowen et al, 1980;Gundelfinger, 1983;Horikoshi et al, 1983;Chuang & Chuang, 1987;Keller & Maniatis, 1991).…”