Cleavage, but not read-through, stimulation activity is responsible for three biologic functions of transcription elongation factor S-II.
Transcription elongation factor S-II stimulates cleavage of nascent transcripts generated by RNA polymerase II stalled at transcription arrest sites. In vitro experiments have shown that this action promotes RNA polymerase II to read through these transcription arrest sites. This S-II-mediated cleavage is thought to be necessary, but not sufficient, to promote read-through in the in vitro systems. Therefore, Saccharomyces cerevisiae strains expressing S-II mutant proteins with different in vitro activities were used to study both the cleavage and the read-through stimulation activities of S-II to determine which S-II functions are responsible for its biologic functions. Strains expressing mutant S-II proteins active in both cleavage and read-through stimulation were as resistant as wild type strains to 6-azauracil and mycophenolic acid. 6-Azauracil also induced IMD2 gene expression in both these mutant strains and the wild type. Furthermore, strains having a genotype consisting of one of these S-II mutations and the spt4 null mutation grew as well as the spt4 null mutant at 37 degrees C, a restrictive temperature for a strain bearing double null mutations of spt4 and S-II. In contrast, strains bearing S-II mutations defective in both cleavage and read-through stimulation had phenotypes similar to those of an S-II null mutant. However, one strain expressing a mutant S-II protein active only in cleavage stimulation had a phenotype similar to that of the wild type strain. These results suggest that cleavage, but not read-through, stimulation activity is responsible for all three biologic functions of S-II (i.e. suppression of 6-azauracil sensitivity, induction of the IMD2 gene, and suppression of temperature sensitivity of spt4 null mutant).
Ubukata
Toshiharu
T
Frontier Project 3, Proteome Research Laboratory, Daiichi Pharmaceutical Company, Ltd., 519 Shimo-Ishibashi, Ishibashi-machi, Shimotsuga-gun, Tochigi 329-0512, Japan.
Shimizu
Tomoko
T
Adachi
Nobuaki
N
Sekimizu
Kazuhisa
K
Nakanishi
Toshiyuki
T
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D005800:Genes, Fungal; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D035165:Transcription Factors, General / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D035602:Transcriptional Elongation Factors
D017931:DNA Primers; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II