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Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection
Author(s) -
Danner Stefan,
Soppa Jörg
Publication year - 1996
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1996.tb02471.x
Subject(s) - biology , haloferax volcanii , dihydrofolate reductase , promoter , microbiology and biotechnology , gene , untranslated region , saturated mutagenesis , consensus sequence , genetics , messenger rna , gene expression , peptide sequence , mutant , archaea
Summary The sequence and spacing requirements of the archaeal‘distal promoter element’(DPE) were examined by randomizing positions ‐19 to ‐32 upstream of the transcriptional start site of the ferredoxin ( fdx ) promoter of Halobacterium salinarium . This randomized promoter library containing 4 14 entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii . Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high‐activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: ‐32 RG TWWWWR AC Y GSY ‐19 (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)‐long transcript, and very resistant clones contained both the 700 nt‐long transcript and a second, more abundant, 500 nt‐long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3’‐untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.