Characterization of the core promoter of the Na+/K+-ATPase alpha 1 subunit gene - Elements required for transcription by RNA polymerase II and RNA polymerase III in vitro

We have analyzed the core promoter element of the Na+/K+-ATPase alpha 1 subunit gene by means of an in vitro transcription system composed of a HeLa nuclear extract. 5'-deletion and 3'-deletion analyses revealed that this gene is specifically transcribed by RNA polymerase II in a manner that is dependent on the upstream regulatory region of the gene (-102 to -61), and that the 3' boundary of the minimal promoter element does not extend beyond +5. Analysis of linker-substitution mutations and point mutations revealed that the TATA-like sequence (-33 to -26) is required for upstream-sequence-dependent transcription whereas linker-substitution mutations and point mutations near +1 did not abolish transcription. The gene was found to be transcribed by RNA polymerase III when phosphocellulose column fractions were assayed. Deletion analysis mapped the minimal RNA-polymerase-III-specific promoter element from -49 to +17. The phosphocellulose 0.3-M-KCl fraction is absolutely required for transcription by RNA polymerase III, while the 0.85-M-KCl fraction represses aberrant transcription from incorrect initiation sites. Analysis of linker-substitution mutations indicated that the TATA-like sequence is required for RNA-polymerase-III-specific transcription. Although point mutations in the 5' half of the TATA-like sequence did not affect transcription, those in the 3' half shifted the transcription initiation site 3 bp upstream. The results suggest that the Na+/K+-ATPase al subunit gene promoter contains a TATA-like sequence which can direct transcription by RNA polymerase III in vitro. The mechanism of alternative regulation of RNA polymerase II and RNA polymerase III is discussed.