Negative elongation factor (NELF) and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) are involved in pausing RNA Polymerase II (Pol II) in the promoter-proximal region of the hsp70 gene in Drosophila, before heat shock induction. Such blocks in elongation are widespread in the Drosophila genome. However, the mechanism by which DSIF and NELF participate in setting up the paused Pol II remains unclear. We analyzed the interactions among DSIF, NELF, and a reconstituted Drosophila Pol II elongation complex to gain insight into the mechanism of pausing. Our results show that DSIF and NELF require a nascent transcript longer than 18 nt to stably associate with the Pol II elongation complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. Taken together, these results provide a possible model by which DSIF binds the elongation complex via association with the nascent transcript and subsequently recruits NELF. Although DSIF and NELF were both required for inhibition of transcription, we did not detect a NELF-RNA contact when the nascent transcript was between 22 and 31 nt long, which encompasses the region where promoter-proximal pausing occurs on many genes in Drosophila. This raises the possibility that RNA binding by NELF is not necessary in promoter-proximal pausing.transcriptional pausing | gene expression | negative elongation factors

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Background: Multiple mechanisms contribute to HIV latency, including NELF-mediated RNA polymerase II (RNAP II) pausing. Results: Paused RNAP II recruits a transcription termination factor and a transcriptional corepressor complex to the HIV promoter. Conclusion: Paused RNAP II couples premature transcription termination and chromatin remodeling to maintain HIV latency. Significance: Paused RNAP II may be targeted to purge latent HIV infection.

Promoter-proximal pausing of RNA polymerase II (Pol II) occurs on thousands of genes in animal cells. This pausing often correlates with the rapid induction of genes, but direct tests of the relationship between pausing and induction rates are lacking. hsp70 and hsp26 in Drosophila are rapidly induced by heat shock. Contrary to current expectations, depletion of negative elongation factor (NELF), a key factor in setting up paused Pol II, reduced pausing but did not interfere with rapid induction. Instead, depletion of NELF delayed the time taken for these genes to shut off during recovery from heat shock. NELF depletion also delayed the dissociation of HSF from hsp70 and hsp26, and a similar delay was observed when cells were depleted of the histone acetyltransferase CBP. CBP has been reported to associate with Pol II, and acetylation of HSF by CBP has been implicated in inhibiting the DNA-binding activity of HSF. We propose that NELF-mediated pausing allows Pol II to direct CBP-mediated acetylation of HSF, thus causing HSF to dissociate from the gene. Activators are typically viewed as controlling Pol II. Our results reveal a possible reciprocal relationship in which paused Pol II influences the activator.Recent analyses of the distribution of RNA polymerase II (Pol II) in animal cells reveal that Pol II is concentrated at the 5Ј end of thousands of genes (11,21,28). In most cases, Pol II is transcriptionally engaged but paused within the first 50 nucleotides of the transcription start site (11,19,24). The pause is mediated by the cooperative activities of DRB sensitivityinducing factor (DSIF) and negative elongation factor (NELF) (43). The duration of the pause varies for different genes, and it is a rate-limiting step in the expression of many genes (17). The kinase P-TEFb appears to control the duration of the pause by phosphorylating Pol II, NELF, and DSIF (8, 34).Possible functions for promoter-proximal pausing are beginning to be identified. Genes that respond rapidly to external signals tend to have paused Pol II (2, 23). The paused state could accelerate the rate of induction by allowing the chromatin remodeling and histone modifications that precede the recruitment of Pol II to be uncoupled from transcription elongation, thus establishing a state that is poised for rapid induction. For some genes, paused Pol II appears to prevent a nucleosome that would otherwise repress transcription initiation from assembling over the core promoter region (18,19). Finally, paused Pol II has been shown to function as an insulator of enhancer function, thus serving as a way to demarcate functional domains in chromatin (10).Promoter-proximal pausing was first identified in the Drosophila hsp70 gene (35). A long-standing hypothesis is that promoter-proximal pausing allows the rapid induction of heat shock genes (25). We set out to test this hypothesis by determining if disrupting promoter-proximal pausing affects the kinetics of induction. RNA interference (RNAi)-mediated depletion of NELF reduces pausing at hsp70 (19, 4...

Gene regulation at the level of translation occurs in response to environmental perturbation and is increasingly recognized as a factor affecting plant development. Despite extensive knowledge of transcriptional control, very little is known about translational regulation of genes in response to the daily light/dark cycles. Here we describe the experimental layout designed to address how the translation states of genes change at various times during a diurnal cycle in Arabidopsis thaliana seedlings. We have adopted a strategy combining sucrose-gradient profiling of ribosomes and high-throughput microarray analysis of the ribosome-associated mRNA to investigate the translational landscape of the Arabidopsis genome. This is a powerful technique that can be easily extended to study translation regulation in different genetic backgrounds and under various environmental conditions.