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 o f a HeLa nuclear extract. 5'-deletion and 3'-deletion analyses reveale d that this gene is specifically transcribed by RNA polymerase II in a manner that is dependent on the upstream regulatory region of the gen e (-102 to -61), and that the 3' boundary of the minimal promoter elem ent does not extend beyond +5. Analysis of linker-substitution mutatio ns and point mutations revealed that the TATA-like sequence (-33 to -2 6) is required for upstream-sequence-dependent transcription whereas l inker-substitution mutations and point mutations near +1 did not aboli sh transcription. The gene was found to be transcribed by RNA polymera se III when phosphocellulose column fractions were assayed. Deletion a nalysis mapped the minimal RNA-polymerase-III-specific promoter elemen t from -49 to +17. The phosphocellulose 0.3-M-KCl fraction is absolute ly required for transcription by RNA polymerase III, while the 0.85-M- KCl fraction represses aberrant transcription from incorrect initiatio n sites. Analysis of linker-substitution mutations indicated that the TATA-like sequence is required for RNA-polymerase-III-specific transcr iption. Although point mutations in the 5' half of the TATA-like seque nce did not affect transcription, those in the 3' half shifted the tra nscription 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 mec hanism of alternative regulation of RNA polymerase II and RNA polymera se III is discussed.