Elements of an archaeal promoter defined by mutational analysis

Abstract

The sequence requirements for specific and efficient transcription from the 16S/23S rRNA promoter of Sulfolobus shibatae were analysed by point mutations and by cassette mutations using an in vitro transcription system. The examination of the box A-containing distal promoter element (DPE) showed the great importance of the TA sequence in the center of box A for transcription efficiency and the influence of the sequence upstream of box A on determining the distance between the DPE and the start site. In most positions of box A, replacement of the wild type bases by adenines or thymines are less detrimental than replacements by cytosines or guanines. The effectiveness of the proximal promoter element (PPE) was not merely determined by its high A + T content but appeared to be directly related to its nucleotide sequence. At the start site a pyrimidine/purine (py/pu) sequence was necessary for unambiguous initiation as shown by analysis of mutants where the wild type start base was replaced. The sequence of box A optimal for promoter function in vitro is identical to the consensus of 84 mapped archaeal promoter sequences.

Gene duplications in evolution of archaeal family B DNA polymerases

“…Putative BoxA motifs, essential for transcription initiation, could be identified in the 5Ј noncoding regions of all four DNA polymerase genes (see Fig. 2) (10,24). S. solfataricus P2 is the first archaeon reported in which three family B DNA polymerases have been found, namely, the two DNA polymerases we report here and the previously sequenced B2 gene (40).…”

Two different respiratory Rieske proteins are expressed in the extreme thermoacidophilic crenarchaeon Sulfolobus acidocaldarius: cloning and sequencing of their genes

“…The pattern of conservation CxHLGC for the first and contain the box A consensus sequence [26]. A pyrimidine-rich CPCHGSxY (x = any amino acid) for the second site is typical stretch of 19 nucleotides containing the sequence TTTTTAT of the membrane bound Rieske proteins of the photosynthetic is located downstream of the stop codon and may act as a and respiratory electron transfer chains rather than of the transcription termination signal [2,27].…”

Transcription of the halophage ΦH repressor gene is abolished by transcription from an inversely oriented lytic promoter

“…In the event, neither the cells carrying the pT64Ko nor those with the pT64LB construct showed significant transcription from the rep promoter ( Fig. 2c and d [19] are underlined. Oligonucleotides used in PCR reactions and primer extension assays are shown under the sequence.…”