Constitutive transcription of yeast ribosomal protein gene TCM1 is promoted by uncommon cis- and trans-acting elements.
The DNA sequence UAST (TCGTTTTGTACGTTTTTCA) was found to mediate transcription of yeast ribosomal protein gene TCM1. UAST was defined as a transcriptional activator on the basis of loss of transcription accompanying deletions of all or part of UAST, orientation-independent restoration of transcription promoted by a synthetic UAST oligomer inserted either into TCM1 or into the yeast CYC1 gene lacking its transcriptional activation region, and diminished transcription following nucleotide alterations in UAST. UAST bound in vitro to a protein denoted TAF (TCM1 activation factor); TAF was concluded to be a transcriptional activator protein because nucleotide alterations in UAST that diminished transcription in vivo also diminished TAF binding in vitro. The sequence of UAST bore no obvious resemblance to UASrpg, the principal cis-acting element common to most yeast ribosomal protein genes. Likewise, TAF was distinguished from the UASrpg-binding protein TUF, since (i) TAF and TUF were chromatographically separable, (ii) binding of either TAF or TUF to its corresponding UAS was unaffected by an excess of UASrpg or UAST DNA, respectively, and (iii) photochemical cross-linking experiments showed that TAF was a protein of 147 kilodaltons (kDa), while TUF was detected as an approximately 120-kDa polypeptide, consistent with its known size. Cross-linking experiments also revealed that both UAST and UASrpg bound a second heretofore unobserved 82-kDa protein; binding of this additional protein appeared to require binding of TAF or TUF. On the basis of the biochemical characterization of TAF and a lack of sequence similarity between UAST and UASrpg, we suggest that transcription of TCM1 is mediated by a cis-acting sequence and at least one trans-acting factor different from the elements which promote transcription of most other ribosomal protein genes. A second trans-acting factor may be shared by TCM1 and other ribosomal protein genes; this factor could mediate coordinate regulation of these genes.
Hamil
K G
KG
Department of Biochemistry, University of North Carolina, Chapel Hill 27599.
Nam
H G
HG
Fried
H M
HM
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology; D005656:Fungal Proteins / Q000502:physiology; D005786:Gene Expression Regulation; D006493:Heparin / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012269:Ribosomal Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012269:Ribosomal Proteins; D014157:Transcription Factors; D006493:Heparin