tailieunhanh - Báo cáo khoa học: Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 andR2mRNAs is restricted to the S-phase of the cell cycle, but the R1 andR2 promoters shownoobvious sequence homologies that could indicate coordination of transcription. Here we study initi-ation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination ofin vivo reporter gene assays andin vitrotranscription. . | Eur. J. Biochem. 270 1791-1801 2003 FEBS 2003 doi Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription Irina Kotova Anna L. Chabes Sergei Lobov Lars Thelander and Stefan Bjorklund Department of Medical Biochemistry and Biophysics Umea University Sweden Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins R1 and R2 which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter which contains an atypical TATA-box with the sequence TTTAAA using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5 -UTR are present the mouse R2 TATA-box is dispensable both for unregulated basal transcription from the R2 promoter and for S-phase specific activity. Instead initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAFns. This interaction down-regulates basal transcription from the R2 promoter both in the absence and in the presence of the TATA-box. Keywords in vitro transcription ribonucleotide reductase TAFs TATA-box transcription regulation. Efficient transcription initiation at a eukaryotic proteinencoding gene requires assembly on promoter DNA of a protein complex containing RNA polymerase II and five general transcription factors GTFs IIB IID IIE IIF and IIH reviewed in 1-3 . TFIID refers to a multiprotein complex composed of the TATA-binding protein TBP and a set of proteins called .