In order to identify previously unknown transcription elongation factors, a genetic screen was carried out to identify mutations that cause lethality when combined with mutations in the genes encoding the elongation factors TFIIS and Spt6. This screen identified a mutation in YKL160W, hereafter named ELF1 (elongation factor 1). Further analysis identified synthetic lethality between an elf1⌬ mutation and mutations in genes encoding several known elongation factors, including Spt4, Spt5, Spt6, and members of the Paf1 complex. Genome-wide synthetic lethality studies confirmed that elf1⌬ specifically interacts with mutations in genes affecting transcription elongation. Chromatin immunoprecipitation experiments show that Elf1 is cotranscriptionally recruited over actively transcribed regions and that this association is partially dependent on Spt4 and Spt6. Analysis of elf1⌬ mutants suggests a role for this factor in maintaining proper chromatin structure in regions of active transcription. Finally, purification of Elf1 suggests an association with casein kinase II, previously implicated in roles in transcription. Together, these results suggest an important role for Elf1 in the regulation of transcription elongation.Eukaryotic transcription is a complex process consisting of a series of steps involving initiation, elongation, and termination. Many recent studies have revealed the extent to which these steps are linked to each other, both functionally and physically (54, 71). While transcription initiation has been well studied and many of the fundamental mechanisms have been identified, an understanding of the control of transcription elongation in vivo is less clear. Some understanding of elongation has come from in vitro experiments examining the effects of various factors in modulating the elongation properties of RNA polymerase II (Pol II) (66). In the past few years many studies have also started to address the control of elongation in vivo and have identified many additional factors believed to play a role in this process (3,69). In this paper, we describe a previously unstudied factor, Elf1 of Saccharomyces cerevisiae, that is functionally related to several other elongation factors and complexes, including TFIIS, Spt6, and the Paf1 complex.TFIIS, the first eukaryotic elongation factor identified, was isolated nearly three decades ago based on its ability to induce long transcripts in an in vitro system (62). Further analysis of TFIIS revealed that it helps elongation in vitro by stimulating an RNA transcript cleavage activity of Pol II (13, 23). This activity allows backtracked (arrested) Pol II to cleave the 3Ј end of the nascent transcript, thus realigning the 3Ј end of the transcript with the Pol II catalytic site. Additional evidence suggests that TFIIS can help Pol II overcome a promoterproximal pause (1, 50). Finally, recent evidence has shown that TFIIS is recruited along transcribed open reading frames, further suggesting a general role in elongation (51, 53).Spt6 is one member of a class of elongat...

E ver-increasing interest is being directed to the role of innate processes in the immune system (1). Particular attention has been given recently to the group of genes that includes the natural killer cell (NK) receptors that are encoded at the leukocyte receptor cluster (LRC) locus on human chromosome 19q13.4 and similar receptors on mouse chromosome 7. These genes, which include killer Ig-like receptors (KIR), paired Ig-like receptors, Ig-like transcripts (ILT) [leukocyte inhibitory receptors (LIR) and monocyte inhibitory receptors (MIR)], leukocyte-associated inhibitory receptor genes, and NKp46͞MAR-1 (2-8), constitute a subset of the Ig gene superfamily (IgSF), and at least some members mediate innate recognition (9). The present understanding of LRC genes is based on studies in mammals; however, it is presumed that they arose early in vertebrate phylogeny.On the basis of considerations of both structural specialization and genetic complexity, it is possible that the Ig and T cell antigen receptor (TCR) multigene families, which undergo segmental reorganization and mediate adaptive immunity, arose from innate immune precursor receptors that are not associated with somatic reorganization. The ability of the Ig and TCR subset of the IgSF to recognize a multitude of antigens relies on extensive diversity in their variable (V) regions and is achieved through site-specific rearrangement of V, diversity (D), and joining (J) segmental elements as well as through additional somatic variation and ultimately somatic hypermutation. Within the V region, diversity in both families of antigen receptors is concentrated in complementarity determining regions (CDRs); variation in CDR1 and CDR2 is encoded in the germ line, whereas variation in CDR3 originates somatically. No evidence has been found for structural features or somatic reorganization associated with V regions in known members of the LRC.A third family of diversified V region-containing receptors, termed novel immune-type receptor (NITR) genes, was identified recently in the compact genome of the pufferfish (10). V regions of NITR genes in this species are organized in families, exhibit variation in CDR1 and CDR2, and like Ig and TCR genes encode J regions but neither rearrange nor appear to exhibit other forms of somatic variation (11). In an overall sense, NITRs are structurally similar to certain members of the LRC in that they possess two extracellular Ig domains, a transmembrane region and a cytoplasmic tail, which contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) that function in negative signal transduction pathways. NITRs possess features of both adaptive and innate immune receptors, and their consideration is significant in addressing the evolution of immune function (12).Despite the utility of the pufferfish genome in terms of the initial identification of NITR genes, this model system is limited in terms of further defining the genetics, developmental regulation, cell lineage-specific expression, and function of NITRs. To address these ...

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.TFIIS is the best-characterized transcription elongation factor at the biochemical level, with several defined biochemical activities (53). TFIIS promotes the elongation of arrested RNA polymerase II (Pol II) by stimulating the inherent RNA cleavage activity of Pol II (13,18). This cleavage allows Pol II to recover from arrest by placing the 3Ј end of the nascent mRNA in the Pol II active site. TFIIS has also been shown to bind Pol II and nucleic acids in vitro (2, 57). A recent structural analysis of TFIIS complexed with Pol II suggested a detailed mechanism for TFIIS function consistent with its biochemical activities. In this structure, the C terminus of TFIIS is able to reach deep into the Pol II secondary channel, approaching the catalytic site of the polymerase (20). This proximity of TFIIS to the nascent RNA allows TFIIS to stimulate the endogenous cleavage activity of Pol II, allowing arrested polymerase complexes to restart elongation by realigning the transcript with the polymerase catalytic site.Although TFIIS is well characterized in vitro, its role in vivo is less well understood. Consistent with the results of biochemical experiments, several genetic and molecular studies with Saccharomyces cerevisiae have also indicated a role for this protein in transcription elongation. First, mutations in DST1, the gene that encodes TFIIS, cause sensitivity to 6-azauracil, a compound that reduces intracellular GTP and UTP levels (17). This sensitivity is believed to be conferred by mutations in elongation factors because the mutant strains are no longer able to efficiently complete transcripts when nucleotide pools are decreased (12). Second, mutations in the DST1 gene exhibit genetic interactions with mutations in other S. cerevisiae genes that encode elongation factors, such as SPT4, SPT5, SPT6, SPT16, and RTF1 (9,15,31,36). Third, TFIIS is required for efficient transcription elongation through the lacZ gene when fused to the yeast GAL1 promoter (24). Finally, chromatin immunoprecipitation (ChIP) experiments have suggested that, under stress conditions, such as cold temperatur...

BackgroundMalarial anaemia is characterized by destruction of malaria infected red blood cells and suppression of erythropoiesis. Interleukin 12 (IL12) significantly boosts erythropoietic responses in murine models of malarial anaemia and decreased IL12 levels are associated with severe malarial anaemia (SMA) in children. Based on the biological relevance of IL12 in malaria anaemia, the relationship between genetic polymorphisms of IL12 and its receptors and SMA was examined.MethodsFifty-five tagging single nucleotide polymorphisms covering genes encoding two IL12 subunits, IL12A and IL12B, and its receptors, IL12RB1 and IL12RB2, were examined in a cohort of 913 children residing in Asembo Bay region of western Kenya.ResultsAn increasing copy number of minor variant (C) in IL12A (rs2243140) was significantly associated with a decreased risk of SMA (P = 0.006; risk ratio, 0.52 for carrying one copy of allele C and 0.28 for two copies). Individuals possessing two copies of a rare variant (C) in IL12RB1 (rs429774) also appeared to be strongly protective against SMA (P = 0.00005; risk ratio, 0.18). In addition, children homozygous for another rare allele (T) in IL12A (rs22431348) were associated with reduced risk of severe anaemia (SA) (P = 0.004; risk ratio, 0.69) and of severe anaemia with any parasitaemia (SAP) (P = 0.004; risk ratio, 0.66). In contrast, AG genotype for another variant in IL12RB1 (rs383483) was associated with susceptibility to high-density parasitaemia (HDP) (P = 0.003; risk ratio, 1.21).ConclusionsThis study has shown strong associations between polymorphisms in the genes of IL12A and IL12RB1 and protection from SMA in Kenyan children, suggesting that human genetic variants of IL12 related genes may significantly contribute to the development of anaemia in malaria patients.