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Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.T ranscription of genes by RNA polymerase II (RNAPII) is a well-orchestrated process that involves steps of initiation, elongation, and termination. Following promoter clearance, RNAPII enters the phase of productive elongation that is achieved by a Brownian ratchet mechanism, in which the RNAPII oscillates between a pretranslocated and a posttranslocated state. After nucleotide addition to the 3= end of the RNA, the incoming nucleoside triphosphate (NTP) locks RNAPII in a posttranslocated form, readying it for the next cycle (1-4). However, productive elongation is not a product of efficient addition of nucleotides by RNAPII alone. During transcription elongation, RNAPII encounters several blocks, including sequence-specific pause sites, nucleotide limitations, DNA lesions, negative elongation factors, and DNA-bound proteins, which cause RNAPII to pause, arrest, or terminate transcription (5, 6). A myriad of elongation factors helps rescue paused/arrested polymerases and stimulate transcription (7,8). Each elongation factor acts via a different mechanism, and often, one works in combination with others.One of the best-characterized elongation factors known to rescue backtracked RNAPII is TFIIS. TFIIS promotes transcription elongation by stimulating the nucleolytic activity of RNAPII and realigning the 3= end of the transcript in the active site of arrested RNAPII (for reviews, see references 9 and 10). New evidence suggests that the cleavage-promoting activity of TFIIS is not the only way in which it stimulates elongation (11-13). Biochemical and biophysical studies have only recently begun to uncover the mechanisms by which TFIIS functions with TFII...

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Cellulosic ethanol has been identified as a crucial biofuel resource due to its sustainability and abundance of cellulose feedstocks. However, current methods to obtain glucose from lignocellulosic biomass are ineffective due to recalcitrance of plant biomass. Insects have evolved endogenous and symbiotic enzymes to efficiently use lignocellulosic material as a source of metabolic glucose. Even though traditional biochemical methods have been used to identify and characterize these enzymes, the advancement of genomic and proteomic research tools are expected to allow new insights into insect digestion of cellulose. This information is highly relevant to the design of improved industrial processes of biofuel production and to identify potential new targets for development of insecticides. This review describes the diverse methodologies used to detect, quantify, purify, clone and express cellulolytic enzymes from insects, as well as their advantages and limitations.