Stabilization of promoter complexes with a single ribonucleoside triphosphate.

Under specific binding conditions RNA polymerase forms complexes at several sites of the replicative form DNA of bacteriophage fd. One of these complexes becomes stable to both high salt and low temperature after incubation with GTP. None of the complexes is stabilized by ATP. The stabilization by GTP results from the synthesis of an oligo(G) chain, which is bound in the complex. Size and pyrimidine fingerprints of the DNA segment protected by the enzyme against digestion with DNase are not changed upon initiation of oligo(G) synthesis. This result indicates that binding site and initiation site are identical parts of a promoter region.     

Single RNA Polymerase Binding Site Isolated

One approach to study the structure of promoter regions^1–3 is to isolate RNA polymerase binding sites. Several attempts have been made to isolate such sites as protected DNA fragments^4–10, but so far the DNA isolated has not definitely shown to be only one or a few specific sites. We report here the isolation of a single RNA polymerase binding site from the replicative form (RF) DNA of bacteriophage fd. The site as isolated is a short double-stranded DNA with a unique nucleotide sequence.     

Studies on bacteriophage fd DNA. III. Nucleotide sequence preceding the RNA start-site on a promoter-containing fragment.

A short DNA fragment containing a strong promoter was isolated from phage fd replicative form DNA with the use of restriction endonucleases, and the sequence of 110 nucleotides in the region preceding the RNA start-site was determined. The sequence was : (5') CGGTCTGGTTCGCTTTGAGGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATCGAATTCGCTTTGCTTCTGACTATAATAGACAGG (3').     

DNA regions essential for the function of a bacteriophage fd promoter.

The promoter for the major coat protein gene of bacteriophage fd contains a unique sequence. TATAAT, in the non-transcribed region corresponding to the Pribnow box. A R-Hha I cleavage site which destroys functions is located five pairs upstream from the TATAAT sequence (fifteen base pairs upstream from the RNA initiation site). The promoter was cleaved into two fragments by R-Hha I and each promoter fragment was joined to DNA fragments derived from other regions. Ligation of the TATAAT-containing fragment to any of the DNA fragments examined resulted in recovery of promoter function. The results suggest for this type of promoter that no unique sequence is necessary upstream from the R-Hha I cleavage site although a contiguous DNA chain must be present in this area.     

Effect of tandem repeated AUG codons on translation efficiency of eukaryotic mRNA carrying a short leader sequence

The effect on translation of multiple copies of the initiation codon AUG at the initiation site in a eukaryotic mRNA carrying a short leader sequence was tested in translation experiments in vitro. DNA, corresponding to a chimeric mRNA sequence consisting of the 5 leader region of brome mosaic virus (BMV) RNA4 and the goat pre--lactalbumin mRNA sequence, was prepared and transcribed in vitro using SP6 RNA polymerase. Site-directed mutagenesis was carried out to change the sequence around the initiation codon AUG. In a wheat germ translation system, the yield of protein obtained using the mRNA with a duplication of the AUG codons at the initiation site was 1.6 times that achieved when only one AUG was present. The rate of formation of the 80S initiation complex was measured by the ribosome binding assay using cycloheximide. A good correlation was observed between the ability to form the complex and translation efficiency.