Transcriptional elongation of the rat apolipoprotein A-I gene: identification and mapping of two arrest sites and their signals.
Previous studies have shown that the elongation phase of apoA-I gene transcription is regulated and contributes to hormone-induced changes in the expression of this gene in rat liver. We have now identified, by in vitro transcription studies with HeLa nuclear extracts, two transcriptional arrest sites within exon 3 and intron 3, respectively. Two truncated transcripts of 510 and approximately 1100 nucleotides in length, termed attenuator 1 RNA and attenuator 2 RNA, respectively, were observed when a rat apoA-I genomic fragment extending from -309 to +1842 relative to the transcription start site was transcribed in vitro in the presence of KCl or Sarkosyl. The attenuation events were promoter-independent as transcription of the apoA-I gene driven by the cytomegalovirus promoter resulted in transcriptional arrest at both sites. Transcription studies using deletion constructs as templates identified nucleotides +976 to +1158 as a region that contained the signal for transcriptional arrest at attenuator site 2. Computational analysis predicted a stem;-loop structure in the nascent RNA immediately upstream of the arrest site. Deletion of attenuator 2 signal or deletion of sequences +147 to +216 located far upstream of the actual elongation block site 1 abrogated arrest at site 1. Thus, complex long-range interactions may be involved in the transcriptional arrest at site 1. These elongation blocks identified in vitro are consistent with earlier in vivo data based on nuclear run-on assays and represent, to our knowledge, the first example describing transcriptional attenuation as a mechanism controlling the expression of a member of the apolipoprotein gene family.
Department of Laboratory Medicine, Landeskrankenanstalten Salzburg, Salzburg, Austria.
G
G
Dallinger
H
H
Oberkofler
C
C
Seelos
W
W
Patsch
D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics*; D001483:Base Sequence; D002474:Cell-Free System; D002874:Chromosome Mapping; D004247:DNA / Q000737:chemistry; D018983:DNA Footprinting; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry; D051381:Rats; D014158:Transcription, Genetic
D016632:Apolipoprotein A-I; D012313:RNA; D004247:DNA