Novel upstream and intragenic control elements for the RNA polymerase III-dependent transcription of human 7SL RNA genes.
In the human nuclear genome only a few copies coding for full-length 7SL RNA genes exist. The Hs7SL-1 gene has recently been classified as type 4 of RNA polymerase III (pol III)-transcribed genes as it was demonstrated that mutations in an external transcriptional activator (ATF) binding site and in an internal CG dinucleotide at positions +15/+16 reduced 7SL RNA expression in vivo and in vitro. We have extended the elucidation of external and internal promoter elements and have discovered two novel regulatory sequences: a TATA-like element in the upstream region and internal A and B box-like motifs. This study was greatly facilitated by the identification of a second, new functional human 7SL RNA gene which we called Hs7SL-3. Remarkably, Hs7SL-3 RNA is synthesized twice as efficiently as Hs7SL-1 in HeLa nuclear extract. Comparison of the upstream regions revealed the presence of two conserved elements in the two human 7SL RNA genes, an ATF/CRE binding site at -43 to -50 and a TATA-like box centered around position -25. Mutational analyses indicated that both external promoter elements are important for efficient transcription. In addition, two sequence motifs can be identified in Hs7SL-1 and Hs7SL-3 at positions 10-19 and 50-60, respectively, downstream of the transcription start site that resemble putative A and B boxes. Single and multiple nucleotide substitutions in these regions also influenced transcription activity to a great extent. The requirement of intragenic functional A and B boxes in combination with the external ATF/CRE and TATA-like promoter elements for the efficient transcription of human 7SL RNA genes is reminiscent of at least two other classes of pol III-transcribed genes in human cells, such as Epstein-Barr virus-encoded EBER and vault RNA genes.
Englert
Markus
M
Institut für Biochemie, Universität Würzburg, Biozentrum, Am Hubland, D-97074 Würzburg, Germany.
Felis
Martha
M
Junker
Volker
V
Beier
Hildburg
H
D024506:5' Flanking Region; D001483:Base Sequence; D001665:Binding Sites; D002457:Cell Extracts; D002883:Chromosomes, Human, Pair 14; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D030541:Databases, Genetic; D005784:Gene Amplification; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012320:RNA Polymerase III / Q000378:metabolism*; D020733:RNA, Small Cytoplasmic / Q000235:genetics*; D018271:Signal Recognition Particle / Q000235:genetics*; D016385:TATA Box; D013698:Templates, Genetic; D014158:Transcription, Genetic
C115990:7SL RNA; D002457:Cell Extracts; D018076:DNA, Complementary; D020733:RNA, Small Cytoplasmic; D018271:Signal Recognition Particle; D012320:RNA Polymerase III