Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.

Abstract

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.Transcription of the Acanthamoeba castellanii rRNA gene in vitro requires a single transcription initiation factor (TIF) and RNA polymerase I (1,14,17). The properties of this system broadly resemble those of other eucaryotic genes (3,8,24) in that TIF functions by forming a stable complex with the promoter and directs RNA polymerase I to accurately initiate transcription (1,14). The promoter of the Acanthamoeba rRNA gene is relatively simple, consisting of an A+T-rich sequence around the transcription start site, which is required for polymerase function, and a second region extending from -20 to -47 (14, 17). This second region is required for TIF binding and can be divided into two portions. One, roughly between -32 and -47, affects the stability of TIF-DNA interactions, and the other, between -31 and -20, is absolutely required for transcription (14,17 (20,23).In previous reports, various aspects of the initiation complex were described (1, 2, 14, 15, 16). In the work described in this report, we combined several approaches to study more dynamic aspects of preinitiation, initiation, and elongating complexes. The results indicate that transcription initiation is accompanied by conformational changes in the polymerase-DNA complex as the enzyme translocates.These changes lead to the conversion of an apparently closed complex to an open complex. Polymerase becomes spatially separated from promoter-bound TIF and covers an unwound DNA region of at least 10 base pairs. MATERIALS AND METHODSProteins and templates. TIF and RNA polymerase I were prepared from A. castellanii as described previously (1). For some experiments, both TIF and RNA polymerase I were further purified on 10 to 30% sucrose gradients to remove contaminating topoisomerase or catenase activities. Following sedimentation, RNA polymerase I was essentially homogeneous, but TIF still contained several bands on sodium dodecyl sulfate gels. Escherichia coli RNA polymerase was the kind gift of A. Y. Woody of our department. The enzyme was more than 95% pure and contained 90% hol...

Photocross-linking of the RNA Polymerase I Preinitiation and Immediate Postinitiation Complexes

“…The A. castellanii TIF-IB complex can be supershifted in an EMSA and forms not one but two supershifted bands, which we interpreted as the binding of one and two immunoglobulins, respectively (19). There is a single MPE-accessible site at the putative dyad (Ϫ49/Ϫ50) of such a dimer (48). A TBP dimer would be immune to inhibition by TATA oligonucleotides, as is found in the pol I system, because the dimer forms by mutual interaction between the TATA-binding surfaces of the two TBP molecules (see Ref.…”

Structural analysis of ternary complexes of vaccinia RNA polymerase.

“…A site of Cu-phenanthroline cleavage at about + 10 of the nontemplate strand of the G21 and G34 templates appeared to be protected in the context of the ternary DEP, which modifies adenosine residues preferentially at unpaired positions in duplex DNA, was used to probe the vaccinia elongation complex paused on the G21 DNA (Fig. 6); this approach had been applied previously to analysis of RNA polymerase I transcription complexes (17). We observed increased reactivity to DEP at three specific adenosine residues (+14, +17, and +20) on the nontemplate strand of the vaccinia ternary complex; two reactive adenosine residues (at +10 and +11) were detected on the template strand.…”

The Fundamental Ribosomal RNA Transcription Initiation Factor-IB (TIF-IB, SL1, Factor D) Binds to the rRNA Core Promoter Primarily by Minor Groove Contacts

“…Ten fmol of TIF-IB in HEG 10 buffer (20 mM HEPES, pH 7.5, 0.1 mM EDTA, and 10% glycerol) or buffer alone was added to the reaction and incubated for 20 min at 25°C. Conditions for the UO 2 (NO 3 ) 2 cutting reactions and precipitations were modified from published procedures (41,42 (43,44). After the addition of 30 l of 1 mg/ml proteinase K and 0.1% SDS solution, samples were incubated at 50°C for 30 min.…”