?(Fig

?(Fig.6ECG).6ECG). chromosomes. We find that Spt5 and Spt6 localize extensively with the phosphorylated, actively elongating form of Pol II, to transcriptionally active sites during salivary gland development and upon heat shock. Furthermore, Spt5 and Spt6 do not colocalize widely with the unphosphorylated, nonelongating form of Pol II. These results strongly suggest that Spt5 and Spt6 play closely related roles associated with active transcription in vivo. mutations cause decreased levels of particular mRNAs (Compagnone-Post and Osley 1996; Hartzog et al. 1998). Under certain in vitro conditions, human Spt4/Spt5 can stimulate the elongation rate of Pol II (Wada et al. 1998a). In addition, Spt4/Spt5, as well as human Spt6 have been implicated in aiding Tat activation of HIV transcription (Wu-Baer et al. 1998; Kim et al. 1999; Parada and Roeder 1999; Ivanov et al. 2000). Finally, much work has shown that P-TEFb is also crucial for Tat activation (for review, see Jones 1997; Garber and Jones 1999). Therefore the in vivo relationship between P-TEFb, Spt4/Spt5, and Spt6 in the regulation of transcription is usually complex, as Spt4/Spt5 appears both to Triisopropylsilane cooperate with and be antagonized by P-TEFb activity, whereas the role of Spt6 is usually less well elucidated in these contexts. Several fundamental aspects of Spt4/Spt5 and Spt6 function are not known. For example, although Spt5 and Spt6 are essential for growth in and have examined the relationship between Spt5, Spt6, and Pol II transcription on polytene chromosomes. Our results show Triisopropylsilane that Spt5 and Spt6 colocalize on polytene chromosomes at a large number of sites. Furthermore, their localization is usually highly coincident with the localization of elongating, phosphorylated Pol II, suggesting that Spt5 and Spt6 are present at most or all regions of active transcription. Consistent with these findings, Spt5, Spt6, and the P-TEFb subunit cyclin T are recruited to heat shock genes induced by heat shock. We also show that a subset of polytene loci, some of which include the highly transcribed Spt5 and Spt6, we obtained cDNAs encoding proteins highly homologous to the human and murine Spt5 and Spt6 proteins. For Spt5 we obtained a full-length cDNA and for Spt6 we deduced the full-length cDNA sequence from overlapping partial cDNAs. Spt4 (polytene map position 49B) and Spt6 (polytene map position 5E) have also been identified by Chiang et al. (1999) and Spt5 has been identified by the Berkeley Genome Project as CG7626 (polytene map position 56D5-6). The predicted proteins are conserved throughout with their corresponding homologs (Fig. ?(Fig.1).1). Among previously noted motifs, the homology between Spt5 proteins from other organisms and the bacterial elongation factor NusG (Hartzog et al. 1998; Wada et al. 1998a; Wu-Baer et al. 1998) is usually conserved in Spt5. For Spt6, all other identified homologs are predicted to have a nonsequence specific DNA binding domain name (HhH domain name) (Doherty et al. 1996) and all but Spt6 have been noted to have an RNA binding domain name (S1 domain name) (Bycroft et al. 1997); these motifs are conserved in Spt6. Open in a separate window Physique 1 Conservation and domain name structure of the Spt5 and Spt6 proteins. Overall regions of homology indicated by dashed lines. (Spt5 domain name structure illustrated with murine Spt5 as a comparison. Spt5 proteins have acidic amino termini (region B), sequence homology to NusG (region C) (Hartzog et al. 1998; Wada et al. 1998a; Wu-Baer et al. 1998), and serine-, threonine-, and proline-rich carboxy-terminal repeat regions noted in Yamaguchi et al. (1999b) and defined as CTR1 and CTR2 in Stachora et al. (1997) (regions D and E). Region E of Spt5 has characteristics of CTR1 and CTR2 but the repeats appear degenerate. Homology of Spt5 determined by BLAST (Altschul et al. 1990) with murine Spt5 is usually 50% amino-acid identity (E value?=?0.0) and with is 26% amino-acid identity (E value?=?1e-58). The amino-terminal RS domain name of (region A) is novel for the Spt5 proteins. (Spt6 domain name structure illustrated with murine Spt6 as a comparison. Like Spt5 proteins, Spt6 proteins have acidic amino-termini (region A). Spt6 proteins also have sequence homology with a prokaryotic family of proteins implicated in transcription regulation (region B, named after the Tex protein (Fuchs et al. 1996) and this region contains a conserved helixChairpinChelix fold (HhH, region C, [Doherty et al. 1996]). Most Spt6 proteins are also predicted to contain RNA-binding S1 domains (region D, [Bycroft et al. 1997]). All Spt6 proteins except Spt6 have extended, divergent carboxyl termini rich in certain amino acids such as serine, threonine, and glycine (Spt6 with murine Spt6 is usually 48% amino-acid identity (E value?=?0.0) and Spt6 is 22% amino-acid identity (E value?=?3e-79). In addition to the conserved regions, Spt5 and Spt6 have regions not found in Triisopropylsilane their homologs. Spt5 is the only known Spt5 homolog that RNF23 contains a stretch of.