Upstream activating sequence

About: Upstream activating sequence is a research topic. Over the lifetime, 1633 publications have been published within this topic receiving 100112 citations.

Mutation analysis of Saccharomyces cerevisiae CDC6 promoter: defining its UAS domain and cell cycle regulating element.

Abstract: Using β-galactosidase as the reporter gene, we carried out mutagenesis experiments to investigate the 5′ promoter region of the CDC6 gene. Our results showed that the DNA element, between −262 and −170, is important for the upstream activating sequence (UAS) activities. On the basis of the DNA sequence, there is a Mlu I (−204) and a Mlu I-like (−216) element located within the middle of the UAS region. Insertion and deletion mutagenesis analysis of the Mlu I sequence has indicated that the internal CGCG sequence of the Mlu I site (ACGCGT) is important for gene expression. Furthermore, when DNA elements containing the Mlu I sites were subcloned into the tester plasmid, periodic expression of a reporter gene throughout the cell cycle was observed, as evidenced by the β-galactosidase activities and lacZ mRNA. Because the possible transcriptional initiation sites of the CDC6 transcript have been previously defined (Zhou and Jong, 1990, J. Biol. Chem. 264, 9022–9029), we propose a model regarding the ...

The chicken delta 1-crystallin gene promoter: binding of transcription factor(s) to the upstream G+C-rich region is necessary for promoter function in vitro

Abstract: There are two linked delta-crystallin genes in the chicken (5' delta 1-delta 2 3'). Only the delta 1 gene has been shown definitively to be active in the lens. Transcription of deletion mutants, reported here, shows that the sequences necessary for the functioning of the delta 1 promoter in a HeLa cell extract are located upstream from the RNA initiation site, between nucleotide positions -121 and -38. This region includes a number of G+C-rich motifs, including one hexanucleotide sequence, CCGCCC, that is repeated six times in the simian virus 40 (SV40) promoter. Competition experiments with purified fragments from the delta 1-crystallin gene promoter showed that binding of transcription factor(s) from the HeLa cell extract to this G+C-rich region is required for promoter activity in vitro. Further, competition experiments using three different fragments from the SV40 promoter suggest that the transcription factor(s) is similar to Sp1, which stimulates transcription by binding to the G+C-rich 21-base-pair repeats of the SV40 promoter, and differs from that which interacts with the SV40 enhancer region.

Characterization of the C operon transcript of bacteriophage Mu.

Abstract: Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.

Cell-free transcription of the nifH1 gene of Methanococcus thermolithotrophicus indicates that promoters of archaeal nif genes share basic features with the methanogen consensus promoter.

Abstract: The nifH1 gene of Methanococcus thermolithotrophicus, which encodes the putative dinitrogenase reductase of an archaeon, was accurately transcribed in a homologous cell-free transcription system. Extracts of cells grown with N2 or ammonia as nitrogen source initiated transcription at the nifH1 promoter with similar efficiencies. We confirmed that cells grown under non-N2-fixing conditions do not contain significant amounts of nifH1-specific mRNA. The levels of cell-free transcription initiation at the nifH1 promoter wer similar to those observed at a tRNA promoter. The DNA sequence from −40 to +5 relative to the initiator nucleotide of nifH1 mRNA contained all the information required for promoter activity. A mutational analysis of this section of DNA demonstrated that a TATA box at −25 and the TTGT motif (initiator element) at the transcription start site are essential for cell-free transcription. These elements are similar to the structural determinants of a known tRNA promoter of Methanococcus. Mutation of a sequence, showing homology to the bacterial NifA site, which overlaps the transcription start site, did not affect promoter activity. Hence, cell-free transcription of the Methanococcus nifH1 gene is independent of upstream activator elements and does not require alternate cis-acting sequences that differ from the methanogen consensus promoter. These findings suggest that the activation of nif promoters is brought about by fundamentally different mechanisms in Archaea and bacteria.