Contact points between transcription machinery and the fibroin gene promoter deduced by functional tests of single-base substitution mutants.

Abstract

An efficient method for oligonucleotide-directed mutagenesis was developed to construct a set of sitespecific mutations by using a mixture of oligodeoxyribonucleotides. With this method, as high as 40% of the tested clones turned out to be desired mutants. Seven single-point mutants were isolated in the "TATA" box region of the fibroin gene. In vitro transcription experiments showed that single-base transversions at the TATA box (A -> T at position -29, T --A or G at -28, A --T at -27, and A --T at -26) resulted in decreased promoter activities, whereas A -+ G transitions at positions -24 and -23 had no effect. The initiation site of transcription was normal in three down-promoter mutants at positions -28 and -26, but the A -* T transversions at positions -29 and -27 induced an additional transcription start from position +4. Using 10 single-point mutants obtained as above or by nitrous acid-induced mutagenesis, we have prepared a pair of heteroduplex DNAs consisting of a mutant strand and the wild-type one. The molecules heterozygous at positions -30, -21, and -20 showed reduced transcription activities when the noncoding strand bears the mutation, whereas that at position -26 gave a low activity when the coding strand carries the mutation. Both types of heteroduplex at positions -29, -28, -27, and -17 exhibited decreased activities. These results suggest a transcription machinery contact to a major groove of the DNA helix at the TATA box and region of position -20.Transcriptional analysis of eukaryotic protein-coding genes has revealed a set of promoter elements (1-11). Core promoter consists of the "TATA" box and its flanking sequence, which is required for fixing the start site as well as efficient transcription. Another element, located at a variable position upstream from the TATA box, appears to modulate the utilization of the core promoter. We believe it is important to fully characterize these elements in order to explore underlying mechanisms of gene expression in eukaryotes. In an attempt to pursue such an approach, we have isolated single-base transition mutants by nitrous acid-induced mutagenesis within the fibroin gene core promoter and have shown that not only the TATA box but also the sequence around position -20 are important for faithful and efficient transcription in vitro (9). However, whether the sequence between the TATA box and the -20 region is essential or not was left unknown because no mutant was available in this area.In study reported here, we used a modified method of oligonucleotide-directed mutagenesis to produce a series of defined point mutants at or near the TATA box of the fibroin gene. Using these mutants, we demonstrated (i) the presence of a silent space between the TATA box and the -20 region substitutable by other bases and (it) the role of the second and the fourth bases of the TATA box in pinpointing the start site. As a next step, we asked which strand of the DNA helix is responsible for the promoter function. For this purpose, we constructed heteroduplex templ...

Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function

“…A threefold reduction in promoter activity in vivo by the inactivation of the NF-1 motif is also in agreement with that reported for two human Pys, BK and JC, and for the murine alpha-l(I) collagen gene (Deyerel & Subramani, 1988;Kumar et al, 1993;Nehls e t al., 1991). Mutagenesis of the TATA box also resulted in a reduction of efficiency of the adenovirus major late promoter and the fibrin gene promoter in vitro by about two-to fivefold (Concino et aI., 1983 ;Hirose et al, 1984). This is also in agreement with the data presented here.…”

The free sulfhydryl group (Cys341) of carboxypeptidase Y: Functional effects of mutational substitutions

“…DISCUSSION 4.1. Oligonucleotide directed semi-random mutagenesis Semi-random mutagenesis by the simultaneous use of several synthetic oligonucleotides may be the preferred method in cases where the effects of of the mutations cannot be predicted (7,19,22). In the present case alteration of the Cys341 codon of CPD-Y, TGT, to a number of different hydrophilic amino acid codons could be achieved by changing this codon to ZAZ, where Z was either C, A or G, thus coding for histidine, glutamine, aspartate, glutamate, asparagine or lysine.…”

Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

“…Another sequence also required for transcription in vitro (11,46,61,89) but dispensible for expression in vivo (4) is the GoldbergHogness box or TATA box located between positions -20 and -30 (M. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1978). Mutation of the TATA box leads to reduced synthesis of RNA in vitro (16,44,45,89). Characterization of several mutants bearing deletions between the TATA box and the cap site as well as mutants which lack the TATA box have led to the conclusion that the TATA box is * Corresponding author.…”