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.

Decreased apoptosis and increased expression of adhesion molecules were noted in OSA PMNs. Although adhesion molecules may facilitate increased PMN-endothelium interactions, decreased apoptosis may further augment these interactions and facilitate free radical and proteolytic enzyme release.

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We tested the hypothesis that in long-term culture conditions, some neutrophils remain viable and participate in debris clearance, and autophagy is involved in their prolonged survival. Neutrophils, classified as professional phagocytes, have the shortest half-life among leukocytes and are constitutively committed to apoptosis. Apoptotic neutrophils are actively removed by Mφ/DCs. However, early and acute inflammatory infiltrates primarily consist of neutrophils. Recently, neutrophils were suggested to facilitate debris clearance at inflammatory sites when the Mφ/DC system is insufficient. Here, purified CD15(+)/CD66b(+)/CD63(+) neutrophils were followed up to 7 days in culture using light, time-lapse, and confocal microscopy. After 3 days in culture, Annexin-V(-)/LC3B(+) large vacuolated cells, engulfing cellular residues, were noted among apoptotic neutrophils and cell debris. Thereafter, these cells were vastly enlarged and exhibited a neutrophilic phenotype (CD15(+)/CD63(+)/MPO(+)/CD66b(+)), phagocytosis, and oxidative burst activity. They also expressed CD68 scavenger receptors and internalized oxLDL. But, unlike in fresh neutrophils or cultured monocytes, oxLDL treatment increased their ROS production. Additionally, these phagocytes contained LC3B-coated vacuoles and LC3B aggregates, indicating the activation of autophagy. An intensive LC3B accumulation was also noted during oxLDL internalization. Importantly, the inhibition of autophagy by 3-MA or BafA1 prevented their development. In conclusion, the internalization of neutrophil remnants may induce activation of autophagic mechanisms in some neutrophil subsets or precursors. This may lead to cell adaptation and survival, resulting in their transformation into long-lived Gφ and potentially suggesting their involvement in inflammatory/anti-inflammatory processes.

Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent inhibitors ofnormal transcription. (2)(3)(4) and the RNAP inhibitor and antibiotic rifampicin (Rif) (5-18).Rif acts by blocking of the initiation-to-elongation transition (9-12). The drug binds to the RNAP-promoter complex and inhibits extension of RNA beyond the initial one or two phosphodiester bonds, but it does not act on elongating complexes. Thus Rif may act in the "vicinity" of the active center, an intuitive locality where the substrate, the product, and the template are held together in appropriate orientation. Mutations conferring resistance to Rif define several clustered residues in the central and amino-terminal parts of the ( polypeptide that are probably involved in Rif binding (5)(6)(7)(8).Other residues contacting nucleotides in the active center were identified by chemical crosslinking and genetic mapping in the carboxyl-terminal part of (3 (Lys-1065 and His-1237) and in the central region of (' (2-4) Rif-Cn-Br compounds. To 0.5 ,umol of aminoalkyl-Rif