Natalia Komissarova

RNA Polymerase Switches between Inactivated and Activated States By Translocating Back and Forth along the DNA and the RNA

Important regulatory events in both prokaryotic and eukaryotic transcription are currently explained in terms of an inchworming model of elongation. In this model, RNA extension is carried out by a mobile catalytic center that, at certain DNA sites, advances within stationary RNA polymerase. This idea emerged from the observation that footprints of individual elongation complexes, haltedin vitro at consecutive DNA positions, can remain fixed on the template for several contiguous nucleotide additions. Here, we examine in detail the structural transitions that occur immediately after the enzyme stops at sites where discontinuous advancement of RNA polymerase is observed. We demonstrate that halting at such special sites does not “freeze” RNA polymerase at one location but induces it to leave its initial position and to slide backward along the DNA and the RNA without degrading the transcript. The resulting loss of contact between the RNA 3′-hydroxyl and the enzyme’s catalytic center leads to temporary loss of the catalytic activity. This process is equilibrated with enzyme return to the original location, so that RNA polymerase is envisaged as an oscillating object switching between catalytically active and inactive states. The retreated isoform constitutes a principal intermediate in factor-induced endonucleolytic RNA cleavage. These oscillations of RNA polymerase can explain its apparent discontinuous advancement, which had been interpreted as indicating flexibility within the enzyme.

Shortening of RNA:DNA Hybrid in the Elongation Complex of RNA Polymerase Is a Prerequisite for Transcription Termination

Passage of E. coli RNA polymerase through an intrinsic transcription terminator, which encodes an RNA hairpin followed by a stretch of uridine residues, results in quick dissociation of the elongation complex. We show that folding of the hairpin disrupts the three upstream base pairs of the 8 bp RNA:DNA hybrid, a major stability determinant in the complex. Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex. During termination, the hairpin does not directly compete for base pairing with the 8 bp hybrid. Thus, melting of the hybrid seems to result from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem. Our results suggest that a similar mechanism disrupts elongation complexes of yeast RNA polymerase II in vitro.

Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase.

Publisher Summary This chapter discusses the use of histidine (His) tags for obtaining transcription intermediates. The mechanism and regulation of transcription depends largely on the development of experimental techniques permitting dissection of the multistep transcriptional cycle. The His tag technology has been applied for the study of mechanisms of elongation, pausing, factor-independent termination, and interaction of RNA polymerase (RNAP) with transcriptional factors. In addition, histidine tags have been used for the rapid purification of RNAP from cells, in vitro reconstitution of RNAP from individually expressed subunits, screening of genetically engineered RNAP mutations, identification of specific fragments among the products of partial proteolysis, and probing of the surface of a RNAP molecule. The chapter discusses the preparation of His-tagged RNAP from cells. Solid-phase transcription with His-tagged RNAP is also given.

Engineering of elongation complexes of bacterial and yeast RNA polymerases

Publisher Summary This chapter discusses the elongation of RNA, accompanied by stepwise forward translocation of RNA polymerase (RNAP) along the template. To understand the mechanisms by which DNA sequences and protein factors regulate elongation, the need to identify the components of the elongation complex (EC) that are targeted by regulatory signals is required. This chapter describes posttranscriptional modifications of the RNA in the EC, utilized to introduce changes in the RNA without altering the sequence of the DNA template and to address the effects of transcript composition on EC stability and catalytic activity. Another set of experimental techniques is based on the reconstitution (assembly) of the EC from RNAP and synthetic RNA and DNA oligonucleotides. This approach is useful for the introduction of mismatches in the RNA, and for obtaining ECs with RNAs shorter than 8 nt. Experiments with assembled ECs allows to address the specific roles of the RNA and the DNA within the regions protected by the enzyme, which could not be accomplished using promoter-initiated ECs.

Assays and affinity purification of biotinylated and nonbiotinylated forms of double-tagged core RNA polymerase II from Saccharomyces cerevisiae.

Publisher Summary This chapter reviews the Pol II core enzyme which has been isolated, using a combination of ion-exchange chromatography and immunoaffinity chromatography with an antibody against the Rpb1 subunit. The alternative to the immunoaffinity approach is introduction of an affinity tag. This strategy was applied successfully to mammalian Pol II, where a FLAG epitope was inserted in the Rpb9 subunit, which allowed achieving a 1000-fold purification in one step. The chapter introduces hexahistidine and biotin acceptor peptide tags in the amino terminus of the Rpb3 subunit of yeast Pol II. According to the Pol II core enzyme and elongation complex crystal structures, tags are located on a surface of the enzyme opposite from the active center and sites of interaction with the DNA and RNA. The tagged enzyme is, indeed, indistinguishable from the wild-type Pol II in the in vitro activity test, and tagged Rpb3 rescues the rpb3 temperature-sensitive phenotype as efficiently as wild-type Rpb3. The chapter also highlights that an alternative protocol, which includes three chromatographic steps, was developed for the purification of nonbiotinylated histidine-tagged Pol II.

Transcription Termination: Primary Intermediates and Secondary Adducts

In living organisms, stable elongation complexes of RNA polymerase dissociate at specific template positions in a process of transcription termination. It has been suggested that the dissociation is not the immediate cause of termination but is preceded by catalytic inactivation of the elongation complex. In vitro reducing ionic strength can be used to stabilize very unstable and catalytically inactive complex at the point of termination; the previous biochemical characterization of this complex has led to important conclusions regarding termination mechanism. Here we analyze in detail the complexes formed between DNA template, nascent RNA, and Escherichia coli RNA polymerase during transcription through the tR2 terminator of bacteriophage λ. At low ionic strength, the majority of elongation complexes fall apart upon reaching the terminator. Released RNA and DNA efficiently rebind RNA polymerase (RNAP) and form binary RNAP·RNA and RNAP·DNA complexes, which are indistinguishable from binary complexes obtained by direct mixing of the purified nucleic acids and the enzyme. A small fraction of elongation complexes that reach termination point escapes dissociation because RNA polymerase has backtracked from the terminator to an upstream DNA position. Thus, transcription elongation to a terminator site produces no termination intermediates that withstand dissociation in the time scale appropriate for biochemical studies.

RNA Folding in Transcription Elongation Complex IMPLICATION FOR TRANSCRIPTION TERMINATION

Intrinsic transcription termination signal in DNA consists of a short inverted repeat followed by a T-rich stretch. Transcription of this sequence by RNA polymerase (RNAP) results in formation of a “termination hairpin” (TH) in the nascent RNA and in rapid dissociation of the transcription elongation complex (EC) at termination points located 7–8 nt downstream of the base of TH stem. RNAP envelops 15 nt of the RNA following RNA growing 3′-end, suggesting that folding of the TH is impeded by a tight protein environment when RNAP reaches the termination points. To monitor TH folding under this constraint, we halted Escherichia coli ECs at various distances downstream from a TH and treated them with single-strand specific RNase T1. The EC interfered with TH formation when halted at 6, 7, and 8, but not 9, nt downstream from the base of the potential stem. Thus, immediately before termination, the downstream arm of the TH is protected from complementary interactions with the upstream arm. This protection makes TH folding extremely sensitive to the sequence context, because the upstream arm easily engages in competing interactions with the rest of the nascent RNA. We demonstrate that by de-synchronizing TH formation and transcription of the termination points, this subtle competition significantly affects the efficiency of transcription termination. This finding can explain previous puzzling observations that sequences far upstream of the TH or point mutations in the terminator that preserve TH stability affect termination. These results can help understand other time sensitive co-transcriptional processes in pro- and eukaryotes.

Iodine-125 radioprobing of E. coli RNA polymerase transcription elongation complexes

Publisher Summary This chapter discusses the Iodine-125 Radioprobing of E. coli RNA polymerase transcription Elongation Complexes. Radioprobing of the Escherichia coli ( E. coli) RNA polymerase (RNAP) elongation complex demonstrate that approximately 9 bp upstream from the active site of RNAP, the DNA/RNA hybrid undergoes a drastic conformational change, and the two strands become separated. This result is in agreement with the crystal structure of the Pol II elongation complex, where the heteroduplex is 9 bp long, and the crystal/crosslinking-based model for the bacterial elongation complex, where the length of the heteroduplex is assumed to be 8-9 bp. The breaks distribution presented for the EC9 and EC10 is similar to that observed by the same method for the EC7 in the case of T7 RNAP. This non-A-like distribution of the breaks reflects the local separation of the RNA and the DNA T-strand. Therefore, the chapter concludes that the DNA/RNA hybrid formed inside the E. coli RNAP is 1-2 bp longer than the hybrid inside the T7 RNAP. The advantage of radioprobing over other foot printing and crosslinking methods is the ability of the DNA/RNA breaking agent—Auger electron—to freely penetrate proteins and nucleic acids.