The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 Å resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3′ end of the RNA in the nucleotide addition site. The 3′ end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5′-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of “switches” at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein–nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, “abortive cycling” during transcription initiation, and RNA and DNA translocation during transcription elongation.

Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions.Accumulating evidence indicates that the expression of many protein coding genes is regulated at the level of transcription elongation. Understanding of elongation control by RNAPII has been hampered by slow progress in the identification of factors involved in transcription elongation. An emerging model is that the interplay of positive and negative elongation factors determines the elongation potential of RNAPII 1 in different promoters (1). Support for this view comes from the recent identification of the negative elongation factors 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivityinducing factor, negative elongation factor, and factor 2 and the positive elongation factor P-TEFb. Factor 2 is an ATP-dependent termination factor that releases transcripts associated with stalled RNAPII molecules (2). DSIF is a repressor of elongation that was identified as a factor that renders in vitro transcription reactions sensitive to the drug DRB (3). NELF works in conjunction with DSIF to repress RNA polymerase II elongation (4). P-TEFb is a DRB-sensitive kinase that is believed to stimulate the elongation potential of RNAPII by phosphorylating the CTD of RNAPII molecules that are engaged in early transcription elongation (5, 6). It was recently suggested that P-TEFb-mediated phosphorylation of the CTD prevents the association of DSIF with RNAPII and thereby overcomes DSIFdependent repression (7). Although it has been long accepted that CTD phosphorylation plays a critical role in transcription, it has been difficult to ascertain the mammalian kinases responsible for CTD phosphorylation in vivo. The observation that the ability of several drugs to block CTD phosphorylation in vivo correlates with the ability of these compounds to inhibit P-TEFb in vitro strongly suggests that P-TEFb might indeed function as a CTD kinase in vivo (8). Additional evidence showing that PTEFb kinase functions as a positive elongation factor in vivo comes from studies with the HIV Tat protein that have shown that the catalytic activity of P-TEFb is required for Tat-dependent stimulation of tra...

Bacteriophage T4 gene 45 protein (gp45) and Escherichia coli beta are DNA‐tracking sliding‐clamp proteins that increase processivity by tethering their conjugate DNA polymerases to DNA. gp45 also activates T4 late transcription. DNA loading of gp45 and beta requires ATP or dATP hydrolysis; efficient loading at primer‐template junctions is assisted by single‐stranded DNA‐binding proteins. The kinetics of gp45 loading and tracking have been examined by DNase I footprinting of linear DNA with one blunt end, one primer‐template junction, and binding sites for proteins that block gp45 tracking. DNA loading of gp45 can also be interrupted by adding the non‐hydrolyzable ATP analog ATP‐gamma‐S. At saturation, DNA is very closely packed with gp45 or beta. When gp45 loading is interrupted, or when a segment of the track is blocked off, the gp45 footprint dissipates within seconds, but the DNA‐tracking state of beta is much more stable. The stability of the tracking state of gp45 is, however, increased by the macromolecular crowding agent polyethylene glycol. We suggest that labile gp45 catenation directly generates the coupling of late transcription to DNA replication during bacteriophage T4 multiplication.

DNA double-crossover molecules containing two Holliday junctions have been prepared and treated with endonuclease VII, the resolvase from bacteriophage T4. One molecule contains antiparallel double-helical domains, and the other molecule contains parallel domains. The parallel double-crossover model system has been made tractable by closing the free ends of the molecule, to convert it to a catenane. The products resulting from the two substrates differ substantially. The molecule containing antiparallel helical domains is cleaved three nucleotides 3' to the crossover points, in a fashion similar to single Holliday junction analogs. The molecule containing parallel helical domains is cleaved, but the major points of scission are five nucleotides 5' to a branch point on the crossover strands and six nucleotides 3' to the same branch point on the non-crossover strands. The major sites of scission reflect features of molecular symmetry in each case, suggesting that the resolvase recognizes structural features. The cleavage results suggest that the antiparallel structure is the natural substrate, if the Holliday junction is unconstrained within the cell. It is straightforward to reconcile antiparallel Holliday junctions with the conventional parallel paradigm of recombination. Nevertheless, the cleavage of the parallel molecule shows that a parallel substrate could also be cleaved symmetrically by endonuclease VII (but with different products) if the molecule were constrained to assume that conformation within the cell.

USA 2Corresponding authorsBacteriophage T4 gene 45 protein, gp45, serves as the sliding clamp of viral DNA replication and as the activator of T4 late gene transcription. In the latter context, DNA tracking is an essential feature of the unique mechanism of action. T4 late promoters, which consist of a simple TATA box, TATAAATA, are recognized by the small a-family gene 55 protein, gp55, which binds to Escherichia coli RNA polymerase core. A direct and RNA polymerase-independent interaction of gp45 with gp55 has been demonstrated in two ways. (i) gp45 tracks along DNA; co-tracking of gp55 requires the previously documented DNA-loading process of gp45, and can be detected by photochemical crosslinking. (ii) The dynamics of DNA tracking by gp45 can be followed by footprinting; the catenated DNAtracking state of gp45 is short-lived, but is stabilized by gp55. The ability of this topologically linked DNAtracking transcriptional activator to interact directly with a promoter recognition protein suggests the existence of multiple pathways of promoter location, which are discussed.