We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

DRB is a classic inhibitor of transcription elongation by RNA polymerase II (pol II). Since DRB generally affects class II genes, factors involved in this process must play fundamental roles in pol II elongation. Recently, two elongation factors essential for DRB action were identified, namely DSIF and P-TEFb. Here we describe the identification and purification from HeLa nuclear extract of a third protein factor required for DRB-sensitive transcription. This factor, termed negative elongation factor (NELF), cooperates with DSIF and strongly represses pol II elongation. This repression is reversed by P-TEFb-dependent phosphorylation of the pol II C-terminal domain. NELF is composed of five polypeptides, the smallest of which is identical to RD, a putative RNA-binding protein of unknown function. This study reveals a molecular mechanism for DRB action and a regulatory network of positive and negative elongation factors.

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.

The elongation step of RNA polymerase II (RNAPII) transcription is emerging as a critical control point for the expression of various genes and for diverse biological processes. Examples include neuronal fate determination during embryonic development (6, 44), gene expression of human immunodeficiency virus (5,11,13,19,43), replication and transcription of hepatitis delta virus (38), and transcriptional regulation of heat shock genes (1,10,18). In all these cases, the involvement of three transcription elongation factors, namely, DRB (5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF), NELF (negative elongation factor), and positive transcription elongation factor b (P-TEFb), has been demonstrated or implicated.Shortly after the initiation of transcription, RNAPII comes under the negative and positive control of DSIF, NELF, and P-TEFb. DSIF and NELF cause transcriptional pausing through physical association with RNAPII. DSIF binds to RNAPII directly and stably (33, 36). However, this appears to have little effect on the catalytic activity of RNAPII (37). A previous study has pointed out that NELF does not bind substantially to DSIF or RNAPII alone but does bind to the complex of DSIF and RNAPII (40). This association is the likely trigger of transcriptional pausing. Conversely, P-TEFb allows RNAPII to enter the productive elongation phase by preventing the action of DSIF and NELF (27, 37). P-TEFb is the protein kinase whose primary target is thought to be the C-terminal domain (CTD) of RNAPII (26). Most, but not all, evidence suggests that P-TEFb-dependent phosphorylation of the CTD facilitates the release of DSIF and NELF from RNA-PII, thereby reversing the inhibition (3, 24, 37). In theory, such regulation at the elongation step allows for rapid change in mRNA levels and for highly sophisticated control over gene expression when combined with regulation at the (pre)initiation step.The structures and functions of DSIF and P-TEFb have been extensively characterized. Human DSIF is a heterodimer composed of p14 (14 kDa) and p160 (160 kDa), whose Saccharomyces cerevisiae counterparts are Spt4 and Spt5 (7,33). In addition to its role in transcriptional pausing, DSIF has a potential to activate RNAPII elongation. The activation mechanism is not well understood: interaction partners of DSIF other than NELF may be involved (13,14,20,23,28). Spt5 has a highly acidic N-terminal region, multiple copies of the KOW motifs, and a repetitive C-terminal region analogous to the RNAPII CTD (9,25,36). RNAPII interacts with Spt5 through a region encompassing the KOW motifs. KOW motifs are also found in the bacterial transcription elongation factor NusG, which binds to prokaryotic RNA polymerase and controls termination and antitermination (15,17,29). In addition, the extreme C terminus of Spt5 is specifically involved in the transcriptional repression pathway (6). Human P-TEFb is a heterodimer composed of Cdk9 (41 kDa) and one of multiple cyclin subunits T1, T2a, T2b, and K (50 to 90 kDa) (26). The k...

DNA replication and transcription of adenovirus (Ad) have been studied extensively as a model eukaryotic system. The dissection and reconstitution of the cell-free DNA replication system using the Ad DNA terminal protein complex (Ad DNAprot) have revealed the detailed mechanism of Ad genome replication (1-3). The Ad genome is a linear DNA of '36 kbp that contains 55-kDa terminal proteins covalently attached to its 5' ends. Replication of the Ad DNA-prot initiates by a protein-priming mechanism in which the 5' terminal nucleotide of the nascent DNA, dCMP, is linked to the 80-kDa