The structure of E. coli core RNA polymerase (RNAP) has been determined to approximately 23 A resolution by three-dimensional reconstruction from electron micrographs of flattened helical crystals. The structure reveals extensive conformational changes when compared with the previously determined E. coli RNAP holoenzyme structure, but resembles the yeast RNAPII structure. While each of these structures contains a thumb-like projection surrounding a channel 25 A in diameter, the E. coli RNAP holoenzyme thumb defines a deep but open groove on the molecule, whereas the thumb of E. coli core and yeast RNAPII form part of a ring that surrounds the channel. This may define promoter-binding and elongation conformations of RNAP, as E. coli holoenzyme recognizes promoter sites on double-stranded DNA, while both E. coli core and yeast RNAPII are elongating forms of the polymerase and are incapable of promoter recognition.

Transcription elongation factors stimulate the activity of DNA-dependent RNA polymerases by increasing the overall elongation rate and the completion of RNA chains. One group of such factors, which includes Escherichia coli GreA, GreB and eukaryotic SII (TFIIS), acts by inducing hydrolytic cleavage of the transcript within the RNA polymerase, followed by release of the 3'-terminal fragment. Here we report the crystal structure of GreA at 2.2 A resolution. The structure contains an amino-terminal domain consisting of an antiparallel alpha-helical coiled-coil dimer which extends into solution, reminiscent of the coiled coil in seryl-tRNA synthetases. A site near the tip of the coiled-coil 'finger' plays a direct role in the transcript cleavage reaction by contacting the 3'-end of the transcript. The structure exhibits an unusual asymmetric charge distribution which indicates the manner in which GreA interacts with the RNA polymerase elongation complex.

A protein identified as the 158-amino acid product of the greA gene was isolated from Escherichia coli. When added to a halted ternary transcription complex, the GreA protein induced cleavage and removal of the 3' proximal dinucleotide from the nascent RNA. The new 3' terminus generated by the cleavage could be extended into longer transcripts. GreA-mediated cleavage of a transcript appears to permit a ternary complex to resume transcription from a state of indefinite elongation arrest induced by a specific DNA site. The GreA protein tended to interact with RNA polymerase during purification and recycled between RNA polymerase molecules in the course of the in vitro cleavage reaction. Similar biochemical activities have been reported in eukaryotic RNA polymerases, indicating that transcript cleavage and restart of elongation may be a general transcriptional mechanism.RNA polymerase is the key enzyme of gene expression and serves as the ultimate target for a myriad of regulatory mechanisms. The search for factors that modify and regulate basic biochemical reactions of RNA polymerase is central to the study of gene regulation. The greA gene of Escherichia coli has been implicated in some vital aspect of transcription by virtue of its ability to suppress, at high copy number, a temperature-sensitive mutation in the RNA polymerase f3 subunit (1, 2). It was proposed that the greA product, which on the basis of DNA sequence should consist of 158 amino acids, directly interacts with the RNA polymerase molecule. We report here a serendipitous finding that GreA is in fact responsible for the recently discovered biochemical reaction of nascent transcript cleavage.The transcript cleavage reaction first described for E. coli RNA polymerase by Chamberlin and coworkers (3) leads to cleavage and removal of a 3' terminal oligonucleotide from the RNA component of a halted elongation complex. After such cleavage, elongation can resume from the newly generated 3' transcript end ifNTPs are present. Similar reactions have since been found in eukaryotic systems and depend on the elongation factor SII (4, 5). In E. coli, the cleavage activity was proposed to be an intrinsic property of RNA polymerase. We demonstrate here that the cleavage reaction is in fact induced by a transacting factor present as a contaminant in standard preparations of E. coli RNA polymerase. The factor has been purified to apparent homogeneity from cell extracts and identified as the product of the greA gene. The reactions were performed in the spin column buffer (the reaction buffer described above but without NTP) in 10-pA samples containing =0.3 pmol of the ternary complex. The incubation at 37TC was for 10 min unless indicated otherwise. Where indicated, combinations of unlabeled ribonucleoside triphosphates were added to a final concentration of 10 ,uM each, as well as the specified amounts of GreA-containing fractions. After incubation, 10 MATERIALS AND METHODS

The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the highresolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA͞RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel.