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The biochemical pathways that are disrupted in the genesis of sporadic breast cancers remain unclear. Moreover, the present prognosticating markers used to determine the prognosis of node-negative patient leads to probabilistic results, and the eventual clinical course is far from certain. Here we identified the human TREX complex, a multiprotein complex that links transcription elongation to mRNA transport, as culprit of aggressive human breast cancers. We show that whereas p84N5 (called hTREX84) is expressed at very low levels in normal breast epithelial cells, it is highly expressed in breast tumors. Importantly, hTREX84 expression correlates with tumor size and the metastatic state of the tumor progression. Reduction of hTREX84 levels in breast cancer cell lines by small interfering RNA result in inhibition of cellular proliferation and abrogation of mRNA export. These results not only identify hTREX84 as a prognosticator of breast cancer but also delineate human TREX complex as a target for therapeutic drugs against breast cancer.

TFIIS is a transcription elongation factor that has been extensively studied biochemically. Although the in vitro mechanisms by which TFIIS stimulates RNA transcript cleavage and polymerase read-through have been well characterized, its in vivo roles remain unclear. To better understand TFIIS function in vivo, we have examined its role during Gal4-mediated activation of the Saccharomyces cerevisiae GAL1 gene. Surprisingly, TFIIS is strongly associated with the GAL1 upstream activating sequence. In addition, TFIIS recruitment to Gal4-binding sites is dependent on Gal4, SAGA, and Mediator but not on RNA polymerase II (Pol II). The association of TFIIS is also necessary for the optimal recruitment of TATA-binding protein and Pol II to the GAL1 promoter. These results provide strong evidence that TFIIS plays an important role in the initiation of transcription at GAL1 in addition to its well-characterized roles in transcription elongation.

A number of proteins and drugs have been implicated in the process of transcriptional elongation by RNA polymerase (Pol) II, but the factors that govern the elongation rate (nucleotide additions per min) and processivity (nucleotide additions per initiation event) in vivo are poorly understood. Here, we show that a mutation in the Rpb2 subunit of Pol II reduces both the elongation rate and processivity in vivo. In contrast, none of the putative elongation factors tested affect the elongation rate, although mutations in the THO complex and in Spt4 significantly reduce processivity. The drugs 6-azauracil and mycophenolic acid reduce both the elongation rate and processivity, and this processivity defect is aggravated by mutations in Spt4, TFIIS, and CTDK-1. Our results suggest that, in vivo, a reduced rate of Pol II elongation leads to premature dissociation along the chromatin template and that Pol II processivity can be uncoupled from elongation rate.

Histone acetylation is associated with transcriptional activation of many genes. However, the role of acetylation in transcriptional regulation of heat shock protein genes (hsp) still remains an obscure issue. Here we examined the effects of histone deacetylase inhibitors (HDIs), trichostatin A, and sodium butyrate, on changes in acetylation level of core histones and on expression of hsp22 gene in Drosophila melanogaster. The results showed that both HDIs elevated the acetylation level of histone H3. By using the chromatin immunoprecipitation, we located the HDI-induced H3 hyperacetylation at both the promoter and the downstream of RNA polymerase II of the transcribing hsp22 gene. Meanwhile, the elevated acetylation level increased the accessibility of heat shock factor to target cis-acting regulatory sites. We conclude that histone acetylation stimulates the transcription initiation and promotes the transcription elongation, thereby up-regulating both basal and inducible expression of hsp22 in D. melanogaster.

Spt6 is a conserved transcription factor that associates with RNA polymerase II (pol II) during elongation. Spt6 is essential for viability in Saccharomyces cerevisiae and regulates chromatin structure during pol II transcription. Here we present evidence that mutations that impair Spt6, a second elongation factor, Spt4, and pol II can affect 3'-end formation at GAL10. Additional analysis suggests that Spt6 is required for cotranscriptional association of the factor Ctr9, a member of the Paf1 complex, with GAL10 and GAL7, and that Ctr9 association with chromatin 3' of GAL10 is regulated by the GAL10 polyadenylation signal. Overall, these results provide new evidence for a connection between the transcription elongation factor Spt6 and 3'-end formation in vivo.

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. Here, we report that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, we discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction.

Background: Several cellular positive and negative elongation factors are involved in regulating RNA polymerase II processivity during transcription elongation in human cells. In recruiting several of these regulatory factors to the 5' long terminal repeat (LTR) promoter during transcription elongation, HIV-1 modulates replication of its genome in a process mediated by the virus-encoded transactivator Tat. One particular cellular regulatory factor, DSIF subunit human SPT5 (hSpt5), has been implicated in both positively and negatively regulating transcriptional elongation but its role in Tat transactivation in vivo and in HIV-1 replication has not been completely elucidated., Results: To understand the in vivo function of hSpt5 and define its role in Tat transactivation and HIV-1 replication, we used RNA interference (RNAi) to specifically knockdown hSpt5 expression by degrading hSpt5 mRNA. Short-interfering RNA (siRNA) designed to target hSpt5 for RNAi successfully resulted in knockdown of both hSpt5 mRNA and protein levels, and did not significantly affect cell viability. In contrast to hSpt5 knockdown, siRNA-mediated silencing of human mRNA capping enzyme, a functionally important hSpt5-interacting cellular protein, was lethal and showed a significant increase in cell death over the course of the knockdown experiment. In addition, hSpt5 knockdown led to significant decreases in Tat transactivation and inhibited HIV-1 replication, indicating that hSpt5 was required for mediating Tat transactivation and HIV-1 replication., Conclusions: The findings presented here showed that hSpt5 is a bona fide positive regulator of Tat transactivation and HIV-1 replication in vivo. These results also suggest that hSpt5 function in transcription regulation and mRNA capping is essential for a subset of cellular and viral genes and may not be required for global gene expression.

The components of chloroplast translation are similar to those of prokaryotic translation but contain some additional unique features. Proteomic analysis of the Chlamydomonas reinhardtii chloroplast ribosome identified an S1-like protein, plastid-specific ribosomal protein-7 (PSRP-7), as a stoichiometric component of the 30S subunit. Here, we report that PSRP-7 is part of a polyprotein that contains PSRP-7 on its amino end and two translation elongation factor Ts (EF-Ts) domains at the carboxy end. We named this polyprotein PETs (for polyprotein of EF-Ts). Pets is a single-copy gene containing the only chloroplast PSRP-7 and EF-Ts sequences found in the C. reinhardtii genome. The pets precursor transcript undergoes alternative splicing to generate three mRNAs with open reading frames (ORFs) of 1.68, 1.8, and 3 kb. A 110-kD pro-protein is translated from the 3-kb ORF, and the majority of this protein is likely posttranslationally processed into the 65-kD protein PSRP-7 and a 55-kD EF-Ts. PETs homologs are found in Arabidopsis thaliana and rice (Oryza sativa). The conservation of the 110-kD PETs polyprotein in the plant kingdom suggests that PSRP-7 and EF-Ts function together in some aspects of chloroplast translation and that the PETs pro-protein may have a novel function as a whole.

TFIIS, an elongation factor encoded by DST1 in Saccharomyces cerevisiae, stimulates transcript cleavage in arrested RNA polymerase II. Two components of the RNA polymerase II machinery, Med13 (Srb9) and Spt8, were isolated as two-hybrid partners of the conserved TFIIS N-terminal domain. They belong to the Cdk8 module of the Mediator and to a subform of the SAGA co-activator, respectively. Co-immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell-free extracts. spt8Delta and dst1Delta mutants were sensitive to nucleotide-depleting drugs and epistatic to null mutants of the RNA polymerase II subunit Rpb9, suggesting that their elongation defects are mediated by Rpb9. rpb9Delta, spt8Delta and dst1Delta were lethal in cells lacking the Rpb4 subunit. The TFIIS N-terminal domain is also strictly required for viability in rpb4Delta, although it is not needed for binding to RNA polymerase II or for transcript cleavage. It is proposed that TFIIS and the Spt8-containing form of SAGA co-operate to rescue RNA polymerase II from unproductive elongation complexes, and that the Cdk8 module temporarily blocks transcription during transcript cleavage.

Segmental patterning in Drosophila relies on a cascade of transcription factors that subdivide the embryo into successively more precise domains. We have identified a missense mutation (W049) in the gene encoding the transcriptional elongation factor Spt5 (reviewed in ) which, when homozygous in the maternal germ line, leads to defects in segmental patterning of the embryo. W049 alters the C-terminal domain of Spt5 and affects its activity in vitro, impairing its abilities to confer sensitivity to the transcriptional inhibitor DRB and to stimulate transcription at limiting nucleotide concentrations. In vivo, W049 shows locus-specific effects on transcription: expression of gap genes remains wild-type, but striped patterning of the primary pair-rule genes even-skipped and runt is disrupted. even-skipped stripes are broadened in the mutant embryos indicating that Spt5 is likely to be a direct, negative regulator of this target gene. Our results suggest control of transcriptional elongation by repressors contributes to striped gene expression in the embryo. By contrast, expression of heat shock-induced proteins is reduced in the mutant embryos. These results provide genetic evidence for Spt5 function during heat shock induction and demonstrate that Spt5 acts both positively and negatively on transcription in vivo depending on context.

The blockage of transcription elongation by RNA polymerase II (pol II) at a DNA damage site on the transcribed strand triggers a transcription-coupled DNA repair (TCR), which rapidly removes DNA damage on the transcribed strand of the expressed gene and allows the resumption of transcription. To analyze the effect of UV-induced DNA damage on transcription elongation, an in vitro transcription elongation system using pol II and oligo(dC)-tailed templates containing a cyclobutane pyrimidine dimer (CPD) or 6-4 photoproduct (6-4PP) at a specific site was employed. The results showed that pol II incorporated nucleotides opposite the CPD and 6-4PP and then stalled. Pol II formed a stable ternary complex consisting of pol II, the DNA damage template, and the nascent transcript. Furthermore, atomic force microscopy imaging revealed that pol II stalled at the damaged region. These findings may provide the basis for analysis of the initiation step of TCR.

The chromatin configuration of DNA inhibits access by enzymes such as RNA polymerase II. This inhibition is alleviated by FACT, a conserved transcription elongation factor that has been found to reconfigure nucleosomes to allow transit along the DNA by RNA polymerase II, thus facilitating transcription. FACT also reorganizes nucleosomes after the passage of RNA polymerase II, as indicated by the effects of certain FACT mutations. The larger of the two subunits of FACT is Spt16/Cdc68, while the smaller is termed SSRP1 (vertebrates) or Pob3 (budding yeast). The HMG-box domain at the C terminus of SSRP1 is absent from Pob3; the function of this domain for yeast FACT is supplied by the small HMG-box protein Nhp6. In yeast, this "detachable" HMG domain is a general chromatin component, unlike FACT, which is found only in transcribed regions and associated with RNA polymerase II. The several domains of the larger FACT subunit are also likely to have different functions. Genetic studies suggest that FACT mediates nucleosome reorganization along several pathways, and reinforce the notion that protein unfolding and (or) refolding is involved in FACT activity for transcription.

Transcriptional interference with convergent transcription from face-to-face promoters is a potentially important form of gene regulation in all organisms. Using LacZ reporter studies, the mechanism of interference was determined for a pair of face-to-face prokaryotic promoters in which a strong promoter interferes 5.6-fold with a weak promoter, 62 bp away. The promoters were variously rearranged to test different models of interference. Terminating transcription from the strong promoter before it reached the weak promoter dramatically reduced interference, indicating a requirement for the passage of the converging RNAP over the weak promoter. Based on in vitro experiments showing a slow rate of escape for open complexes at the weak promoter and their sensitivity to head-on collisions with elongating RNAP, a "sitting duck" model of interference is proposed and supported with in vivo permanganate footprinting. The model is further supported by the analysis of a second set of prokaryotic face-to-face promoters.

Considerable advances into the basis of RNA-polymerase-II-mediated transcriptional regulation have recently emerged. Biochemical, genetic and structural studies have contributed to novel insights into transcription, as well as the functional significance of covalent histone modifications. New details regarding transcription elongation through chromatin have further defined the mechanism behind this action, and identified how chromatin structure may be maintained after RNAP II traverses a nucleosome. ATP-dependent chromatin remodeling complexes, along with histone chaperone complexes, were recently discovered to facilitate histone exchange. In addition, it has become increasingly clear that transcription by RNA polymerase II extends beyond RNA synthesis, towards a more active role in mRNA maturation, surveillance and export to the cytoplasm.

The yeast Paf1 complex, minimally composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, was originally isolated in association with RNA polymerase II (Pol II). Paf1 complex components are abundant and colocalize with Pol II on chromatin at promoters and in the coding regions of actively transcribed genes. Loss of Paf1 results in severe phenotypes and reduced amounts of other Paf1 factors, with little effect on abundance or chromatin distribution of Pol II, proteins important for transcriptional elongation (Spt5, Spt16), or RNA processing (Sub2). Loss of Paf1 factors causes a reduction of Pol II Ser2 phosphorylation and shortened poly(A) tails, suggesting that the complex facilitates linkage of transcriptional and posttranscriptional events. Surprisingly, loss of Rtf1 or Cdc73, with little phenotypic consequence, results in loss of Paf1 factors from chromatin and a significant reduction in Paf1/Pol II association. Therefore, the major functions of Paf1 can be independent of actively transcribing Pol II.

The kinetochore (centromeric DNA and associated proteins) mediates the attachment of chromosomes to the mitotic spindle apparatus and is required for faithful chromosome transmission. We established that evolutionarily conserved Saccharomyces cerevisiae SPT4, previously identified in genetic screens for defects in chromosome transmission fidelity (ctf), encodes a new structural component of specialized chromatin at kinetochores and heterochromatic loci, with roles in kinetochore function and gene silencing. Using chromatin immunoprecipitation assays (ChIP), we determined that kinetochore proteins Ndc10p, Cac1p, and Hir1p are required for the association of Spt4p to centromeric (CEN) loci. Absence of functional Spt4p leads to altered chromatin structure at the CEN DNA and mislocalization of the mammalian CENP-A homolog Cse4p to noncentromeric loci. Spt4p associates with telomeres (TEL) and HMRa loci in a Sir3p-dependent manner and is required for transcriptional gene silencing. We show that a human homolog of SPT4 (HsSPT4) complements Scspt4-silencing defects and associates with ScCEN DNA in an Ndc10p-dependent manner. Our results highlight the evolutionary conservation of pathways required for genome stability in yeast and humans.

Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.

In eukaryotic cells, genomic DNA is assembled with chromosomal proteins, mainly histones, in a highly compact structure termed chromatin. In this form, DNA is not readily accessible to the cellular machineries, which require DNA as a template. Dynamic changes in chromatin organization play a critical role in regulation of DNA-dependent processes such as transcription, DNA replication, recombination and repair. Chromatin structure is altered in transcriptionally active loci: the basic chromatin unit, the nucleosome, appears to be depleted for one histone H2A/H2B dimer. Previously, reconstitution of RNA polymerase II (PolII)-driven transcription on chromatin templates in a highly purified in vitro system led to identification of FACT (for facilitates chromatin transcription), which was required for productive transcript elongation through nucleosomes. FACT was proposed to promote PolII transcription through nucleosomes by removing either one or both H2A/H2B dimers. Here we present an overview of the earlier studies, which resulted in the initial identification and characterization of FACT, as well as the recent findings that refine the model for the mechanism of FACT function in transcription.

The transcription elongation complex, once thought to be composed of merely the DNA template, RNA polymerase II and the nascent RNA transcript, is now burgeoning as a unit as multifaceted and complicated as the transcription initiation complex. Studies concentrated in defining the elongation stage of transcription during the past recent years have resulted in the discovery of a diverse collection of transcription elongation factors that are either directly involved in the regulation of the rate of the elongating RNA polymerase II or can modulate messenger RNA (mRNA) processing and transport. Such studies have demonstrated that the elongation stage of transcription is highly regulated and has opened a new era of studies defining the molecular role of such transcription elongation factors in cellular development, differentiation and disease progression. Recent studies on the role of RNA polymerase II elongation factors in regulating of the overall rate of transcription both in vitro and in vivo, histone modification by methylation and organismal development will be reviewed here.

TFIIS promotes the intrinsic ability of RNA polymerase II to cleave the 3'-end of the newly synthesized RNA. This stimulatory activity of TFIIS, which is dependent upon Rpb9, facilitates the resumption of transcription elongation when the polymerase stalls or arrests. While TFIIS has a pronounced effect on transcription elongation in vitro, the deletion of DST1 has no major effect on cell viability. In this work we used a genetic approach to increase our knowledge of the role of TFIIS in vivo. We showed that: (1) dst1 and rpb9 mutants have a synthetic growth defective phenotype when combined with fyv4, gim5, htz1, yal011w, ybr231c, soh1, vps71, and vps72 mutants that is exacerbated during germination or at high salt concentrations; (2) TFIIS and Rpb9 are essential when the cells are challenged with microtubule-destabilizing drugs; (3) among the SDO (synthetic with Dst one), SOH1 shows the strongest genetic interaction with DST1; (4) the presence of multiple copies of TAF14, SUA7, GAL11, RTS1, and TYS1 alleviate the growth phenotype of dst1 soh1 mutants; and (5) SRB5 and SIN4 genetically interact with DST1. We propose that TFIIS is required under stress conditions and that TFIIS is important for the transition between initiation and elongation in vivo.

Macronucleus of Tetrahymena divides amitotically, although in a microtubule-dependent fashion. Besides the localization study and pharmacological study of macronuclear microtubules, mechanism of the macronuclear division is poorly understood. A biochemical search for microtubule-associated protein was attempted from the isolated macronucleus. Improvement on macronucleus isolation method and microtubule coprecipitation assay led to the cloning of p138, a new homologue of transcription elongation factor FACT (facilitates chromatin transcription) 140kDa subunit. DNase treatment test of macronuclear extract and the sequence of p138 suggested that p138 is associated with chromosome in the macronucleus. The release tests of p138 from microtubules indicated that p138 is released from microtubules in the presence of ATP but not in the presence of AMP-PNP. Together, the results suggest a novel function of FACT homologue, that p138 interacts with both microtubules and chromosome.

The elongation of transcription is a highly regulated process that requires negative and positive effectors. By binding the double-stranded stem in the transactivation response (TAR) element, RD protein from the negative transcription elongation factor (NELF) inhibits basal transcription from the long terminal repeat of the human immunodeficiency virus type 1 (HIVLTR). Tat and its cellular cofactor, the positive transcription elongation factor b (P-TEFb), overcome this negative effect. Cdk9 in P-TEFb also phosphorylates RD at sites next to its RNA recognition motif. A mutant RD protein that mimics its phosphorylated form no longer binds TAR nor represses HIV transcription. In sharp contrast, a mutant RD protein that cannot be phosphorylated by P-TEFb functions as a dominant-negative effector and inhibits Tat transactivation. These results better define the transition from abortive to productive transcription and thus replication of HIV.

Transcription by RNA polymerase II (pol II) requires the ordered binding of distinct protein complexes to catalyze initiation, elongation, termination, and coupled mRNA processing events. One or more proteins from each complex are known to bind pol II via the carboxy-terminal domain (CTD) of the largest subunit, Rpb1. How binding is coordinated is not known, but it might involve conformational changes in the CTD induced by the Ess1 peptidyl-prolyl cis/trans isomerase. Here, we examined the role of ESS1 in transcription by studying one of its multicopy suppressors, BYE1. We found that Bye1 is a negative regulator of transcription elongation. This led to the finding that Ess1 also inhibits elongation; Ess1 opposes elongation factors Dst1 and Spt4/5, and overexpression of ESS1 makes cells more sensitive to the elongation inhibitor 6-AU. In reporter gene assays, ess1 mutations reduce the ability of elongation-arrest sites to stall polymerase. We also show that Ess1 acts positively in transcription termination, independent of its role in elongation. We propose that Ess1-induced conformational changes attenuate pol II elongation and help coordinate the ordered assembly of protein complexes on the CTD. In this way, Ess1 might regulate the transition between multiple steps of transcription.

While a number of proteins are involved in elongation processes, the mechanism for action of most of these factors remains unclear primarily because of the lack of suitable in vivo model systems. We identified in yeast several genes that contain internal poly(A) sites whose full-length mRNA formation is reduced by mutations in RNA polymerase II subunit RPB2, elongation factor SPT5, or TFIIS. RPB2 and SPT5 defects also promoted the utilization of upstream poly(A) sites for genes that contain multiple 3' poly(A) signaling sequences, supporting a role for elongation in differential poly(A) site choice. Our data suggest that elongation defects cause increased transcriptional pausing or arrest that results in increased utilization of internal or upstream poly(A) sites. Transcriptional pausing or arrest can therefore be visualized in vivo if a gene contains internal poly(A) sites, allowing biochemical and genetic study of the elongation process.

Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1TPAWNSGSK9. We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1Delta mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30 degrees C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine.

Previous studies have suggested that transcription elongation results in changes in chromatin structure. Here we present studies of Saccharomyces cerevisiae Spt6, a conserved protein implicated in both transcription elongation and chromatin structure. Our results show that, surprisingly, an spt6 mutant permits aberrant transcription initiation from within coding regions. Furthermore, transcribed chromatin in the spt6 mutant is hypersensitive to micrococcal nuclease, and this hypersensitivity is suppressed by mutational inactivation of RNA polymerase II. These results suggest that Spt6 plays a critical role in maintaining normal chromatin structure during transcription elongation, thereby repressing transcription initiation from cryptic promoters. Other elongation and chromatin factors, including Spt16 and histone H3, appear to contribute to this control.

The lateral flexible stalk of the large ribosomal subunit is made of several interacting proteins anchored to a conserved region of the 28S (26S) rRNA termed the GTPase-associated domain or thiostrepton loop. This structure is demonstrated to adopt puzzling changes of conformation following the different steps of the elongation cycle. Some of these proteins termed the P-proteins in eukaryotes and L10 and L7/L12 in bacteria, present little structural similarities between Eubacteria on one side and Archae and Eukaryotes on the other side. However, up to now, these proteins seem to present a similar macromolecular organisation and they have been involved in the same functions. Convincing evidence attests that these proteins participate in elongation factor binding to the ribosome, and it has been suggested that these proteins might be evolved in a GTP hydrolysis activating protein activity. Involvement of these proteins in the translational mechanism is discussed. Moreover, in eukaryotes, small P-proteins are also found as isolated proteins in a cytoplasmic pool that exchanges with the ribosome-associated P-proteins. Moreover, a part of the ribosomal proteins is phosphorylated (hence their P-protein names). The biological signification of these particularities is discussed.

The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors, linked to the poor prognosis, being refractory to chemo- and radio-therapies as well as the advanced stage of tumorous lesions in particular. However, both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic. By comparing various upstream segments of the Hsp70 gene for each's ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line, we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression. Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene, but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage for targeting the therapeutic gene expression to human tumors.

Immobilized DNA templates, glycerol gradient centrifugation, and native gel analysis were utilized to isolate and compare functional RNA polymerase II (RNAPII) elongation complexes from Saccharomyces cerevisiae and human cell nuclear extracts. Yeast elongation complexes blocked by incorporation of 3'-O-methyl-GTP into the nascent transcript exhibited a sedimentation coefficient of 35S, were less tightly associated to the template than their human counterparts, and displayed no detectable 3'-5' exonuclease activity on the associated transcript. In contrast, blocked human elongation complexes were more tightly bound to the template, and multiple forms were identified, with the largest exhibiting a sedimentation coefficient of 60S. Analysis of the associated transcripts revealed that a subset of the human elongation complexes exhibited strong 3'-5' exonuclease activity. Although isolated human preinitiation complexes were competent for efficient transcription, their ability to generate 60S elongation complexes was strikingly impaired. These findings demonstrate functional and size differences between S. cerevisiae and human RNAPII elongation complexes and support the view that the formation of mature elongation complexes involves recruitment of nuclear factors after the initiation of transcription.

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.

We have examined the localization and targeting of the RNA polymerase II (pol II) transcription elongation factor TFIIS in amphibian oocyte nuclei by immunofluorescence. Using a novel antibody against Xenopus TFIIS the major sites of immunostaining were found to be Cajal bodies, nuclear organelles that also contain pol II. Small granular structures attached to lampbrush chromosomes were also specifically stained but the transcriptionally active loops were not. Similar localization patterns were found for the newly synthesized myc-tagged TFIIS produced after injection of synthetic transcripts into the cytoplasm. The basis of the rapid and preferential targeting of TFIIS to Cajal bodies was investigated by examining the effects of deletion and site-specific mutations. Multiple regions of TFIIS contributed to efficient targeting including the domain required for its binding to pol II. The localization of TFIIS in Cajal bodies, and in particular the apparent involvement of pol II binding in achieving it, offer further support for a model in which Cajal bodies function in the preassembly of the transcriptional machinery. Although our findings are therefore consistent with TFIIS playing a role in early events of the transcription cycle, they also suggest that this elongation factor is not generally required during transcription in oocytes.

We have previously shown that yeast mutants of the THO complex have a defect in gene expression, observed as an impairment of lacZ transcription. Here we analyze the ability of mutants of different transcription elongation factors to transcribe lacZ. We found that spt4Delta, like THO mutants, impaired transcription of lacZ and of long and GC-rich DNA sequences fused to the GAL1 promoter. Using a newly developed in vitro transcription elongation assay, we show that Spt4 is required in elongation. There is a functional interaction between Spt4 and THO, detected by the lethality or strong gene expression defect and hyper-recombination phenotypes of double mutants in the W303 genetic background. Our results indicate that Spt4-Spt5 has a positive role in transcription elongation and suggest that Spt4-Spt5 and THO act at different steps during mRNA biogenesis.

During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.

The elongation step of transcription by RNA polymerase II (RNAPII) is controlled both positively and negatively by over a dozen cellular proteins. Recent findings suggest that two distinct viruses, human immunodeficiency virus type 1 and hepatitis delta virus, encode proteins that facilitate viral replication and transcription by targeting the same cellular transcription elongation machinery.