Analysis of Gene Induction and Arrest Site Transcription in Yeast with Mutations in the Transcription Elongation Machinery

Abstract

In vitro, transcript elongation by RNA polymerase II is impeded by DNA sequences, DNA-bound proteins, and small ligands. Transcription elongation factor SII (TFIIS) assists RNA polymerase II to transcribe through these obstacles. There is however, little direct evidence that SII-responsive arrest sites function in living cells nor that SII facilitates readthrough in vivo. Saccharomyces cerevisiae strains lacking elongation factor SII and/or containing a point mutation in the second largest subunit of RNA polymerase II, which slows the enzyme's RNA elongation rate, grow slowly and have defects in mRNA metabolism, particularly in the presence of nucleotide-depleting drugs. Here we have examined transcriptional induction in strains lacking SII or containing the slow polymerase mutation. Both mutants and a combined double mutant were defective in induction of GAL1 and ENA1. This was not due to an increase in mRNA degradation and was independent of any drug treatment, although treatment with the nucleotide-depleting drug 6-azauracil exacerbated the effect preferentially in the mutants. These data are consistent with mutants in the Elongator complex, which show slow inductive responses. When a potent in vitro arrest site was transcribed in these strains, there was no perceptible effect upon mRNA accumulation. These data suggest that an alternative elongation surveillance mechanism exists in vivo to overcome arrest. Several factors have been identified that act as transcription elongation factors in vitro.One of the best studied is transcription elongation factor SII (also known as TFIIS), which facilitates readthrough of transcription arrest sites and other blocks to RNA polymerase II (pol II) 1 elongation in vitro (1, 2). SII acts by binding to pol II and activating an intrinsic RNA cleavage activity within the enzyme that shortens the newly transcribed RNA and allows re-extension of the arrested transcript past the block to elongation (1, 2).The transcription arrest process has been difficult to study in vivo, due in part to the rapid processing of primary transcripts and degradation of incomplete transcripts. Sensitivity of yeast to nucleotide-depleting drugs such as 6-azauracil (6AU) and mycophenolic acid has served as a phenotypic indicator of yeast with mutations in genes encoding pol II subunits and transcription elongation factors, including RPB1, RPB2, RPB6, RPB9, DST1, ELP1, ELP3, SPT4, SPT5, SPT6, SPT16,. This drug sensitivity is thought to result from stress upon the elongation machinery due to a reduction in the intracellular pools of nucleotides used for RNA synthesis (3,8). It is well known that, in vitro, a low concentration of nucleotide substrates causes pol II to transcribe at a slower rate, become arrested more often, and therefore be dependent upon SII for efficient elongation (4, 14 -17). Hence, upon drug treatment, yeast may become more dependent upon efficient transcript elongation by pol II. Nevertheless, not all 6AU-sensitive mutants carry mutations in genes that are obviously related to tra...

Interaction between Transcription Elongation Factors and mRNA 3′-End Formation at the Saccharomyces cerevisiae GAL10-GAL7 Locus

“…1) when cells are grown at permissive temperature (30°C). Thus, an spt6 mutation causes defects similar to what has been previously observed for other putative elongation mutants (63)(64)(65)68).…”

In Vivo Evidence that Defects in the Transcriptional Elongation Factors RPB2, TFIIS, and SPT5 Enhance Upstream Poly(A) Site Utilization

“…In particular, the rpb2-10 protein causes arrest at known mammalian pause sites and requires elongation factor TFIIS to overcome the arrest. Moreover, the rpb2-10 allele in combination with a dst1 (encoding TFIIS) deletion causes severe defects in the synthesis of most mRNA in vivo (29) and reduces the induction of a number of other genes (56). These observations suggest that in vivo rpb2-10 actually causes promiscuous arrest of RNA Pol II that requires TFIIS to suppress and overcome.…”

“…For instance, no particular gene whose elongation is affected by these factors has been identified (29,45,56), although it has been suggested that transcription of long genes and genes with high GϩC content is defective with certain elongation defects (9, 43). Similarly, whole genome microarray analysis with dst1 and rpb9 alleles did not yield particular genes controlled at the level of elongation (23).…”

Genetic Interactions of DST1 in Saccharomyces cerevisiae Suggest a Role of TFIIS in the Initiation-Elongation Transition

“…In this sense, a Ca 2ϩ -dependent association of TFIIS with Cmd1/calmodulin has been reported (Stir- agreement with this idea, it has been reported that mRNA levels in general and specifically of ENA1, a gene required for salt tolerance, are severely affected in a actin and tubulin, resistance to killer toxin, and two dst1 background when combined with the rpb2-10 allele ORFs of unknown function. Among the genes uncov- (Lennon et al 1998;Wind-Rotolo and Reines 2001). ered using the SGA approach, HTZ1 and SOH1 particiFunction of the multicopy suppressors of soh1 dst1: pate in RNApol II transcription, GIM5 encodes for a…”

“…1999), speed the polymerization rate up and/or and Young 1995; Myers and Kornberg 2000; Davis et down (Bengal et al 1991;Chafin et al 1991;Hartzog al. 2002), and at some promoters can be recruited beet al 1998;Wada et al 1998), or reactivate arrested fore the loading of the core polymerase (Bhoite et al polymerases concomitantly to transcript cleavage (Wind 2001;Cosma et al 2001;Park et al 2001). RNApol II and Reines 2000).…”