Two hybrid promoters that are functional in Escherichia coli have been constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the first hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the second hybrid promoter (tacII), the DNA upstream of position -11 at the Hpa I site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by two unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter and approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor and both can be derepressed with isopropyl beta-D-thiogalactoside. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.

https://www.pnas.org/content/pnas/80/1/21.full.pdf

The tac promoter: a functional hybrid derived from the trp and lac promoters

Transcriptional control signals of a model eukaryotic protein-coding gene have been identified by a new procedure of in vitro mutagenesis. This method allows small clusters of nucleotide residues to be substituted in a site-directed manner without causing the addition or deletion of other sequences. Transcription assays of a systematic series of these clustered point mutants have led to the identification of three distinct control signals located within the 105-nucleotide residues immediately upstream from the point where transcription begins.

/pdf/transcriptional-control-signals-of-a-eukaryotic-protein-v3s280jg9g.pdf

Transcriptional control signals of a eukaryotic protein-coding gene

In vivo protein-DNA interactions at the developmentally regulated enhancer of the mouse muscle creatine kinase (MCK) gene were examined by a newly developed polymerase chain reaction (PCR) footprinting procedure. This ligation mediated, single-sided PCR technique permits the exponential amplification of an entire sequence ladder. Several footprints were detected in terminally differentiated muscle cells where the MCK gene is actively transcribed. None were observed in myogenic cells prior to differentiation or in nonmuscle cells. Two footprints appear to correspond to sites that can bind the myogenic regulator MyoD1 in vitro, whereas two others represent muscle specific use of apparently general factors. Because MyoD1 is synthesized by undifferentiated myoblasts, these data imply that additional regulatory mechanisms must restrict the interaction between this protein and its target site prior to differentiation.

https://science.sciencemag.org/content/sci/246/4931/780.full.pdf

In vivo footprinting of a muscle specific enhancer by ligation mediated PCR

The mouse heavy chain immunoglobulin gene contains a tissue-specific enhancer. The enhancer and flanking sequences were studied in vivo by carrying out dimethyl sulfate protection experiments on living cells, in combination with genomic sequencing. Relative to reactions on naked DNA, there are changes (protections and enhancements) in the reactivity of guanine residues to dimethyl sulfate within the enhancer sequence in myeloma, B, and early B cells, whereas virtually no alterations appear in cells of non-B lineage. Most of the affected residues are in four clusters, in sequences homologous to the octamer 5'CAGGTGGC 3' (C, cytosine; A, adenine; G. guanine; T, thymine). The alterations in the pattern of G reactivity are consistent with the tissue-specific binding of molecules to the mouse immunoglobulin heavy chain enhancer.

https://science.sciencemag.org/content/sci/227/4683/134.full.pdf

B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo

Structure of the Bacterial RNA Polymerase Promoter Specificity σ Subunit

The chemical alkylating agent dimethyl sulfate can probe the interaction between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and the purine bases of a promoter. This agent methylates the N7 position on guanine or the N3 position on adenine; the bound protein can either protect these positions or affect the reactivity to produce an enhanced methylation. The pattern of DNA residues in the lactose promoter protected from, or enhanced to, methylation by a specifically bound polymerase shows that the enzyme covers a region of at least 38 base pairs, stretching upstream from the origin of transcription. These protein-DNA contacts occur predominantly in the major groove of the DNA helix. Furthermore, this pattern of methylation shows that the polymerase unwinds the helix at the origin of transcription. The relationship between polymerase-DNA contacts defined by dimethyl sulfate and known features of promoter structure is discussed. To facilitate these experiments I have constructed a plasmid that permits a unique 5'-end labeling of each strand of a 95-base-pair fragment containing a lac operon promoter. This plasmid contains two copies of the lac promoter-operator region.

https://www.pnas.org/content/pnas/75/11/5314.full.pdf

Lorraine Johnsrud

Papers

Contacts between Escherichia coli RNA polymerase and a lac operon promoter