NELF and DSIF act together to inhibit transcription elongation in vitro, and are implicated in causing promoter proximal pausing on the hsp70 gene in Drosophila. Here, further characterization of Drosophila NELF is provided. Drosophila NELF has four subunits similar to subunits of human NELF. The amino acid sequences of NELF-B and NELF-D are highly conserved throughout their lengths, while NELF-A and NELF-E contain nonconserved regions inserted between conserved N- and C-terminal regions. Immunodepletion of NELF or DSIF from a nuclear extract desensitizes transcription in vitro to DRB. Immunodepletion of NELF also impairs promoter proximal pausing on the hsp70 promoter in vitro without affecting initiation. Chromatin immunoprecipitation analyses detect NELF at the promoters of the hsp70 and β1-tubulin genes where promoter proximal pausing has been previously detected. Heat shock induction of hsp70 results in a marked decrease in NELF at the hsp70 promoter. Immunofluorescence analysis of polytene chromosomes shows extensive colocalization of the NELF-B and NELF-D subunits at hundreds of interbands. Neither subunit appears to be recruited to puffs. These results provide a foundation for genetic and biochemical analysis of NELF in Drosophila.

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Transcription can be divided into three stages: initiation, elongation, and termination. Initiation involves the binding of RNA polymerase (RNAP) 2 to a gene's promoter and the beginning of RNA synthesis. Elongation involves lengthening the nascent transcript by the addition of nucleotides to the 3Ј-end of the RNA located in the active site of RNAP. The last stage of transcription, termination, involves stopping RNA synthesis, release of the transcript, and dissociation of the RNAP from the DNA template. Termination and Gene RegulationTranscription termination is essential for subdividing and retrieving information from the genomes of all organisms. RNAPII transcribing from an upstream region can interfere with the function of a promoter in its path (1). Termination is also essential for recycling RNAP.Termination has long been known to play a significant role in regulating transcription through genes in bacteria, and isolated cases hint at its potential importance in eukaryotes. In bacteria, attenuation and antitermination are related processes in which mutually exclusive patterns of RNA folding dictate whether the polymerase will continue transcription or prematurely terminate before completing transcription of an operon (2). It is less clear what role premature termination plays in regulating gene expression in eukaryotes. Measurement of the distribution of RNAPII throughout the genome in human cells identifies hundreds of genes in which the density of RNAPII at the 5Ј-end greatly exceeds the density of RNAPII at the 3Ј-end of the gene (3), but in many cases, RNAPII may be stalling as a result of its association with the negative elongation factors NELF and DSIF (4).One case in eukaryotic cells in which premature termination is involved in repressing transcription is the HIV provirus. RNAPII is concentrated at the 5Ј-end of a transcriptionally repressed provirus (5). That this discontinuous distribution of RNAPII is a consequence at least in part of premature termination is indicated by the accumulation of short transcripts in the cytoplasm of the cell (6). Recently, we provided evidence that Pcf11, a protein previously implicated in termination at the ends of protein-encoding genes, could be responsible for premature termination on HIV (7). Activation of HIV transcription involves a protein called Tat. Tat can be thought of as an antiterminator. It functions by recruiting the kinase P-TEFb to the elongation complex (EC). Phosphorylation of RNAPII and other regulators of elongation renders RNAPII less prone to premature termination (4).Corden and co-workers (8) recently uncovered a negative feedback mechanism that could be the first clear example of attenuation of a eukaryotic gene. Nrd1 participates in terminating RNAPII transcription at the ends of small nuclear RNA genes in yeast, and it binds specific sequences near the 3Ј-end of small nuclear RNA (9). Corden and co-workers (8) observed that mutations in the Nrd1 protein cause overexpression of NRD1. Further analysis revealed that there are numerous Nrd...