Primary Transcription Start Site Research Articles

The Initiator Element and Proximal Upstream Sequences Affect Transcriptional Activity and Start Site Selection in the Amyloid β-Protein Precursor Promoter

The TATA-less human amyloid beta-protein precursor promoter contains an initiator element with the sequence CGTCA+1GTT. Primary transcriptional start sites were identified at positions +1 and -4. Deletion of the upstream activator elements APBbeta and APBalpha did not affect the selection of transcriptional start sites, although total transcriptional activity was reduced both in vitro and in vivo. Mutations within the initiator element shifted the transcriptional start sites and reduced transcriptional activity. Mutations between positions -6 and -35 changed the relative utilization of start sites +1 and -4 without affecting the total level of transcriptional activity. A 10-base pair deletion between position -40 and -31 increased in vitro transcriptional activity with a preeminent utilization of the start site at position -4. In contrast, a 20-base pair deletion between position -40 and -21 resulted in a reduction in transcriptional activity and in the primary utilization of the start site at position +1. Furthermore, transactivation by APBbeta and APBalpha was eliminated. DNase I footprinting provided evidence for the existence of two binding domains designated UE (position -12 to -30) and Inr (position +7 to -7). The positions of these binding domains are altered in mutations and deletions that affect transcriptional activity.

Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts.

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.

Identification of primary transcriptional start sites of mouse mitochondrial DNA: accurate in vitro initiation of both heavy- and light-strand transcripts.

The major transcriptional control sequences of vertebrate mitochondrial DNA lie within the displacement loop region. Transcription events initiating in the displacement loop sequence of the mouse genome were identified by 5' end mapping of primary transcripts by S1 nuclease protection and primer extension techniques. Light-strand transcription initiates at a single site, 165 nucleotides upstream of the major heavy-strand origin of replication. Transcription of the heavy strand occurs at two distinct sites, 5 and 13 nucleotides upstream of the gene for phenylalanyl-tRNA, the first heavy-strand-encoded gene. This spatial relationship of the two transcriptional start sites with each other and with the origin of heavy-strand replication and the gene for tRNAPhe is quite similar to that for human mitochondrial DNA. The predominant form of primary heavy-strand transcript in mouse is a short, ca. 75-nucleotide, RNA containing the sequences of tRNAPhe and a few additional nucleotides at the 5' end of tRNAPhe, suggesting that the processing of tRNA involves independent cleavages at the 5' and 3' ends of tRNA sequences.