Caroline M. Kane

Promoting elongation with transcript cleavage stimulatory factors.

Transcript elongation by RNA polymerase is a dynamic process, capable of responding to a number of intrinsic and extrinsic signals. A number of elongation factors have been identified that enhance the rate or efficiency of transcription. One such class of factors facilitates RNA polymerase transcription through blocks to elongation by stimulating the polymerase to cleave the nascent RNA transcript within the elongation complex. These cleavage factors are represented by the Gre factors from prokaryotes, and TFIIS and TFIIS-like factors found in archaea and eukaryotes. High-resolution structures of RNA polymerases and the cleavage factors in conjunction with biochemical investigations and genetic analyses have provided insights into the mechanism of action of these elongation factors. However, there are yet many unanswered questions regarding the regulation of these factors and their effects on target genes.

Purification and Characterization of an RNA Polymerase II Phosphatase from Yeast

RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa.

Transcript elongation factor TFIIS is involved in Arabidopsis seed dormancy.

Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells lacking TFIIS are viable, inactivation of mouse TFIIS causes embryonic lethality. Here, we have identified a protein encoded in the Arabidopsis genome that displays a marked sequence similarity to TFIIS of other organisms, primarily within domains II and III in the C-terminal part of the protein. TFIIS is widely expressed in Arabidopsis, and a green fluorescent protein-TFIIS fusion protein localises specifically to the cell nucleus. When expressed in yeast cells lacking the endogenous TFIIS, Arabidopsis TFIIS partially complements the sensitivity of mutant cells to the nucleotide analog 6-azauridine, which is a typical characteristic of transcript elongation factors. We have characterised Arabidopsis lines harbouring T-DNA insertions in the coding sequence of TFIIS. Plants homozygous for T-DNA insertions are viable, and genomewide transcript profiling revealed that compared to control plants, a relatively small number of genes are differentially expressed in mutant plants. TFIIS(-/-) plants display essentially normal development, but they flower slightly earlier than control plants and show clearly reduced seed dormancy. Plants with RNAi-mediated knockdown of TFIIS expression also are affected in seed dormancy. Therefore, TFIIS plays a critical role in Arabidopsis seed development.

Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex

Elongation complexes of RNA polymerase II, RNA-DNA-enzyme ternary complexes, are intermediates in the synthesis of all eukaryotic mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and its interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyribonuclease I (DNAse I) to map the interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzyme-template interactions of individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformational transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.

Isolation and characterization of the Schizosaccharomyces pombe gene encoding transcript elongation factor TFIIS

A gene designated tfs1 has been isolated from Schizosaccharomyces pombe based on its similarity to genes encoding transcription elongation factor TFIIS. The nucleotide sequence of the tfs1 gene predicts a polypeptide with similarity to mammalian, Drosophila and Saccharomyces cerevisiae TFIIS. A haploid Sz. pombe strain with tfs1 deleted from the genome is viable. Thus, tfs1 is not essential for viability. However, deletion of tfs1 results in slow growth and increased sensitivity to the drug 6‐azauracil, a phenotype similar to that of a S. cerevisiae strain deleted for the gene encoding TFIIS. The DNA sequence of tfs1 has been deposited in GenBank under Accession Number U20526.

Characterization of a HeLa cDNA clone encoding the human SII protein, an elongation factor for RNA polymerase II

We present the cloning and sequence characterization of a HeLa cDNA encoding the SII transcription elongation factor. This cDNA clone is distinct from those previously isolated from a human kidney cDNA library [Yoo et al., Nucleic Acids Res. 19 (1991) 1073-1079]. Southern analysis suggests that more than one gene may exist for SII in the human genome. A comparison of deduced amino acid sequences for SII-related proteins from a variety of eukaryotes demonstrates very high similarity, especially within the C-terminal domain.

Structural Basis for the Species-specific Activity of TFIIS

Many proteins involved in eukaryotic transcription are similar in function and in sequence between organisms. Despite the sequence similarities, there are many factors that do not function across species. For example, transcript elongation factor TFIIS is highly conserved among eukaryotes, and yet the TFIIS protein from Saccharomyces cerevisiae cannot function with mammalian RNA polymerase II and vice versa. To determine the reason for this species specificity, chimeras were constructed linking three structurally independent regions of the TFIIS proteins from yeast and human cells. Two independently folding domains, II and III, have been examined previously using NMR (1-3). Yeast domain II alone is able to bind yeast RNA polymerase II with the same affinity as the full-length TFIIS protein, and this domain was expected to confer the species selectivity. Domain III has previously been shown to be readily exchanged between mammalian and yeast factors. However, the results presented here indicate that domain II is insufficient to confer species selectivity, and a primary determinant lies in a 30-amino acid highly conserved linker region connecting domain II with domain III. These 30 amino acids may physically orient domains II and III to support functional interactions between TFIIS and RNA polymerase II.

Genetic Interactions Between TFIIF and TFIIS

The eukaryotic transcript elongation factor TFIIS is encoded by a nonessential gene, PPR2, in Saccharomyces cerevisiae. Disruptions of PPR2 are lethal in conjunction with a disruption in the nonessential gene TAF14/TFG3. While investigating which of the Taf14p-containing complexes may be responsible for the synthetic lethality between ppr2Δ and taf14Δ, we discovered genetic interactions between PPR2 and both TFG1 and TFG2 encoding the two larger subunits of the TFIIF complex that also contains Taf14p. Mutant alleles of tfg1 or tfg2 that render cells cold sensitive have improved growth at low temperature in the absence of TFIIS. Remarkably, the amino-terminal 130 amino acids of TFIIS, which are dispensable for the known in vitro and in vivo activities of TFIIS, are required to complement the lethality in taf14Δ ppr2Δ cells. Analyses of deletion and chimeric gene constructs of PPR2 implicate contributions by different regions of this N-terminal domain. No strong common phenotypes were identified for the ppr2Δ and taf14Δ strains, implying that the proteins are not functionally redundant. Instead, the absence of Taf14p in the cell appears to create a dependence on an undefined function of TFIIS mediated by its N-terminal region. This region of TFIIS is also at least in part responsible for the deleterious effect of TFIIS on tfg1 or tfg2 cold-sensitive cells. Together, these results suggest a physiologically relevant functional connection between TFIIS and TFIIF.

Identifying regulators of transcript elongation in eukaryotes.

Publisher Summary The elongation step of transcription is a dynamic process that provides many opportunities for regulation. Biochemical analysis of this reaction has allowed transcript elongation to be staged into several distinct mechanistic steps, each of which can be assayed. The assays themselves are simple and technically straightforward, but their design and interpretation require some understanding of the transcript-elongation process. Many eukaryotic genes are subject to some form of regulation during the elongation process. The regulation is influenced by trans-acting factors that can stimulate or inhibit the activity of the transcribing RNA polymerase. This chapter highlights the various methods and the types of assays that can be used to identify such factors in eukaryotic cells. The chapter illustrates the elongation assays of one such factor, TFIIS. The process of transcript elongation is discussed, with a particular emphasis on the definition of some of the commonly used terms and the steps where transcription regulation might occur.

Purification and assay of Saccharomyces cerevisiae phosphatase that acts on the C-terminal domain of the largest subunit of RNA polymerase II.

Publisher Summary This chapter reviews the largest subunit of RNA polymerase II, containing a heptad repeat with the consensus sequence YSPTSPS at its C terminus, referred to as the CTD. The heptad is repeated multiple times, with 26 repeats in saccharomyces cerevisiae and 52 repeats in rodents and humans. The phosphorylation along this repeated sequence varies during transcription, during the cell cycle, and with changes in cellular metabolism. Several kinases have participated in the differential phosphorylation of this repeat, but only one phosphatase that has been isolated is specific for the polymerase. Several assays used to quantitate the phosphatase activity are presented, as are several methods for purification of the phosphatase itself, from the more traditional biochemical method to methods used with recombinant proteins over-expressed in bacterial and insect cells. The method using tandem affinity purification (TAP) to isolate recombinant protein from the native environment of the yeast cells is included in this chapter.