EST1, EST2, EST3 and TLC1 function in a single pathway for telomere replication in the yeast Saccharomyces cerevisiae [1] [2], as would be expected if these genes all encode components of the same complex. Est2p, the reverse transcriptase protein subunit, and TLC1, the templating RNA, are subunits of the catalytic core of yeast telomerase [3] [4] [5]. In contrast, mutations in EST1, EST3 or CDC13 eliminate telomere replication in vivo [1] [6] [7] [8] but are dispensable for in vitro telomerase catalytic activity [2] [9]. Est1p and Cdc13p, as components of telomerase and telomeric chromatin, respectively, cooperate to recruit telomerase to the end of the chromosome [7] [10]. However, Est3p has not yet been biochemically characterized and thus its specific role in telomere replication is unclear. We show here that Est3p is a stable component of the telomerase holoenzyme and furthermore, association of Est3p with the enzyme requires an intact catalytic core. As predicted for a telomerase subunit, fusion of Est3p to the high affinity Cdc13p telomeric DNA binding domain greatly increases access of telomerase to the telomere. Est1p is also tightly associated with telomerase; however, Est1p is capable of forming a stable TLC1-containing complex even in the absence of Est2p or Est3p. Yeast telomerase therefore contains a minimum of three Est proteins for which there is both in vivo and in vitro evidence for their role in telomere replication as subunits of the telomerase complex.

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To identify regions of the largest subunit of RNA polymerase that are potentially involved in transcript elongation and termination, we have characterized amino acid substitutions in the 13' subunit of Escherichia coli RNA polymerase that alter expression of reporter genes preceded by terminators in vivo. Termination-altering substitutions occurred in discrete segments of [3', designated 2, 3a, 3b, 4a, 4b, 4c, and 5, many of which are highly conserved in eukaryotic homologs of 13'. Region 2 substitutions (residues 311-386) are tightly clustered around a short sequence that is similar to a portion of the DNA-binding cleft in E. coli DNA polymerase I. Region 3b (residues 718-798) corresponds to the segment of the largest subunit of RNA polymerase II in which amanitin-resistance substitutions occur. Region 4a substitutions (residues 933-936) occur in a segment thought to contact the transcript 3' end. Region 5 substitutions (residues 1308-1356) are tightly clustered in conserved region H near the carboxyl terminus of [3'. A representative set of mutant RNA polymerases were purified and revealed unexpected variation in percent termination at six different p-independent terminators. Based on the location and properties of these substitutions, we suggest a hypothesis for the relationship of subunits in the transcription complex.[Key Words: rpoC gene; [3' subunit; RNA polymerase; transcriptional termination] Received July 26, 1994; revised version accepted October 13, 1994.Escherichia coli RNA polymerase contains a core of four subunits: 6', 155 kD; [3, 150 kD; and two c~, 43 kD each. These subunits form a scaffold and catalytic center for synthesis of RNA on a double-stranded DNA template that appear to be conserved from bacteria to humans. The two largest subunits, 6' and B in E. coli, display significant sequence similarity to homologous subunits found in all multisubunit RNA polymerases (Allison et al. 1985; Jokerst et al. 1989; Young 1991 and references therein): Each contains eight to nine conserved, colinear segments, although no sequence conservation is evident between f3' and B. However, we currently lack a clear picture of how these conserved primary sequence motifs are positioned in the three-dimensional structure of RNA polymerase.In the transcription elongation complex (for review, see Das 1993;Chamberlin 1994;Chan and Landick 1994), RNA polymerase contacts the DNA over a 25-to ~Present address:

The role of yeast RNA polymerase II (pol II) subunit RPB9 in transcript elongation was investigated by examining the biochemical properties of pol II lacking RPB9 (pol II⌬9). The maximal rate of chain elongation was nearly identical for pol II and pol II⌬9. By contrast, pol II⌬9 elongated more efficiently through DNA sequences that signal the elongation complex to pause or arrest. The addition of purified recombinant RPB9 to pol II⌬9 restored the elongation properties of the mutant polymerase to those of the wild-type enzyme. Arrested pol II⌬9 complexes were refractory to levels of TFIIS that promoted maximal read-through with pol II. However, both pol II and pol II⌬9 complexes stimulated with TFIIS undergo transcript cleavage, confirming that transcript cleavage and read-through activities can be uncoupled. Our observations suggest that both TFIIS and RPB9 are required to stimulate the release of RNA polymerase II from the arrested state.The mRNA transcription machinery in eukaryotes is a complex of more than 30 different polypeptides of which 12 polypeptides are tightly associated with RNA polymerase (pol) 1 II. The largest two subunits of pol II, which are thought to harbor catalytic activity, are related to the largest subunits of the other nuclear RNA polymerases (1, 2). Each of the remaining pol II subunits is conserved throughout the Eukarya and more than half are also conserved in the Archae (3).In Saccharomyces cerevisiae, the genes encoding the twelve subunits of pol II have been cloned and sequenced. Five of the ten small subunits of yeast pol II (RPB5, RPB6, RPB8, RPB10, and RPB12) are common to all three nuclear RNA polymerases (4, 5). Several remaining pol II subunits have homologues in pol I and pol III. RPB11 (6) is related to AC19, a subunit shared by pols I and III (7). RPB7 is similar to the pol III subunit C25 (8). RPB9 is related to the pol I subunit, A12.2 (9). At least six pol II small subunits from S. cerevisiae are functionally interchangeable with human subunits (RPB6, RPB7, RPB8, RPB9, RPB10, and RPB12) (10 -12). Genetic analysis indicates that only two yeast pol II subunits, RPB4 and RPB9, are not essential for cell viability (13,14).In yeast, deletion of RPB9 results in mild temperature sensitivity and relatively normal levels of transcription in vivo. However, for most genes examined, the selectivity of the site of transcription initiation is altered, with new start sites shifted upstream relative to wild-type sites (15-17). The transcription initiation phenotype can be recapitulated in vitro, and addition of recombinant purified RPB9 (rRPB9) restores wild-type start site selection (15). RPB9 from S. cerevisiae is a 122-amino acid polypeptide that contains two zinc binding domains (14). The COOH-terminal zinc binding domain shares 25% sequence identity with that of the general transcript elongation factor TFIIS (18) and is predicted to adopt a zinc ribbon fold (17)(18)(19). This domain is required for the function of RPB9 in start site selection 2 and is required within TFIIS for el...

The Saccharomyces cerevisiae protein Stm1 was originally identified as a G4 quadruplex and purine motif triplex nucleic acid-binding protein. However, more recent studies have suggested a role for Stm1p in processes ranging from antiapoptosis to telomere maintenance. To better understand the biological role of Stm1p and its potential for G*G multiplex binding, we used epitope-tagged protein and immunological methods to identify the subcellular localization and protein and nucleic acid partners of Stm1p in vivo. Indirect immunofluorescence microscopy indicated that Stm1p is primarily a cytoplasmic protein, although a small percentage is also present in the nucleus. Conventional immunoprecipitation found that Stm1p is associated with ribosomal proteins and rRNA. This association was verified by rate zonal separation through sucrose gradients, which showed that Stm1p binds exclusively to mature 80 S ribosomes and polysomes. Chromatin immunoprecipitation experiments found that Stm1p preferentially binds telomere-proximal Y element DNA sequences. Taken together, our data suggest that Stm1p is primarily a ribosome-associated protein, but one that can also interact with DNA, especially subtelomeric sequences. We discuss the implications of our findings in relation to prior genetic, genomic, and proteomic studies that have identified STM1 and/or Stm1p as well as the possible biological role of Stm1p.