RNA polymerase promoter

The first step in creating a specific mRNA species is to construct a plasmid encoding the mRNA behind a T7, T3, or SP6 RNA polymerase promoter sequence. The S boundary of the mRNA is defined by the position of the promoter transcription start site and the S end is defined by restriction digest of the plasmid prior to in vitro transcription. 

Template availability Methylation Exposure of DNA Attraction for RNA polymerase Promoter TATA, CAT Transcription factors Alternative Poly(A) tailing Alternative splicing Alternative translation start... 

The suppressor tRNA developed by the Chamberlin lab for use in a rabbit reticulocyte lysate is based on an E. coli glycyl tRNA, which was initially chosen because glycyl-tRNA synthetases do not rely on a double-sieve editing mechanism for enzymatic hydrolysis of misacylated tRNAs [26]. Two base pair changes were made to the acceptor stem to allow incorporation of the optimal T7 RNA polymerase promoter into the DNA template for tRNA y-Con [27,28],... 

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

Amplify an appropriate DNA fragment with an appropriate polymerase according to the manufacturer s instruction (see Note 1 and 6). Make sure to tag the RNA polymerase promoter site to the 5 end of the reverse primer see Fig. 1). 

At least three types of proteins regulate transcription initiation by RNA polymerase specificity factors alter the specificity of RNA polymerase for a given promoter or set of promoters repressors impede access of RNA polymerase to the promoter and activators enhance the RNA polymerase-promoter interaction. 

As mentioned above, aptamers can also be made of DNA. Their selection (Figure 7.3) differs slightly from an RNA aptamer selection (Figure 7.2). Instead of the in vitro transcription of DNA into RNA, ssDNA is prepared as described in Section 7.3.3.1. Of course, inclusion of an RNA polymerase promoter in the template design, as well as the reverse transcription step, are not necessary. All other steps are the same as those for RNA aptamer selection (see Section 7.3.1). 

Double-stranded DNA template comprising T7 RNA polymerase promoter sequence (S -ACGCACGCTGTAATACGACTCACTATA-3 promoter italicized) and reverse complement of the tagging RNA (5 -yCCCATCACCATCTCTTGCTATAGTGAGTCGTATTAC AGCGTCCGT-S boldface portion complementary to tagging RNA sequence two underlined nucleotides 2 -OMe)... 

The EMBL vector systems have been subjected to many modifications to enhance their usefulness. The most useful modifications involve the addition of more restriction enzyme recognition sites flanking the stuffer fragment and the addition of T3 and T7 RNA polymerase promoters. The T3 and T7 RNA polymerase promoters are used to synthesize 32P-labeled RNAs that can be used as probes for the identification of overlapping A clones. Many of the A vectors described above are available from or have been developed by commercial vendors. As in the case with many basic molecular biology techniques, the components needed to construct a specific recombinant phage library (A vector arms, in vitro packaging extracts, etc.) and even the library can be obtained from commercial sources. 

Rosa, M. D. (1979). Four T7 RNA Polymerase Promoters Contain an Identical 23 Base Pair Sequence. Cell 16 815. 

The tasks of transcriptional and translational signal recognition involve the prediction of promoters and sites that function in the initiation and termination of transcription and translation. Bacterial promoter sites, specifically the Escherichia coli RNA polymerase promoter site, are now very well characterized. The main problem is that the two conserved regions of the bacterial promoter, the -10 and -35 regions, are separated from each other by 15 to 21 bases, making the detection of the entire promoter as a single pattern difficult. Eukaryotic promoters are less well characterized than their bacterial equivalents. The major elements are the CCAAT box, GC box, TATA box and cap site. 

Tabor, S. and Richardson, C. C.(1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078. 

The cDNA encoding the fusion protein is cloned into a plasmid downstream of a SP6, T7 or T3 RNA polymerase promoter. The plasmid is linearized downstream of the inserts by digesting with an appropriate restriction enzyme, and transcribed in vitro with the appropriate RNA polymerase (75). 

Instead of specific amplification of one target to improve sensitivity, methods that amplify all genomic DNA or mRNAs are useful when the target is in short supply. For example, multiple-displacement amplification uses exonuclease-resistant random hexamers and a highly pro-cessive polymerase to amplify DNA nonspecificaily. Initial DNA denaturation is not necessary and the reaction proceeds isothermally. Similarly, messenger RNA can be generi-caUy amplified with a poly(T) primer modified with an RNA polymerase promoter. After reverse transcription, second-strand DNA synthesis, and transcription, antisense RNA is produced. Both whole genome and antisense RNA amplification are also useful as nucleic acid purification methods before amplification or detection. 

G. M. T. Cheetham, D. Jeruzalini, and T. A. Steitz Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature 399, 80 (1999). 

Any plasmid designed to express recombinant proteins in E. coli under the control of a bacteriophage RNA polymerase promoter can be used in an E. coli cell-free system. As a mle of thumb, constructs that express weU in vivo tend to express well in vitro. T7 RNA polymerase is often considered intrinsically more efficient than other RNA polymerases (see Part IV, Chapter 12). However, this differential efficiency can often be attributed to differential sensitivity to salt concentration. Because of the relative robustness and efficiency of the T7 polymerase, plasmids under the control of a T7 promoter are almost exclusively used in E. coli cell-free systems. 

Although housekeeping genes are expressed con-stitutively, the cellular concentrations of the proteins they encode vary widely. For these genes, the RNA polymerase-promoter interaction strongly influences the rate of transcription initiation differences in promoter sequence allow the ceU to synthesize the appropriate level of each housekeeping gene product. 

A. Revyakin, A.N. Kapanidis, W. Niu, Y.W. Ebright, R. Levy, R.H. Ebright, Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex, Cell 2002, 108, 599-614. 

There are a number of approaches for the expression of proteins in E. coli. A modern approach to protein expression is to make use of purpose designed pDNA expression vectors such as the pET vector family, freely available from commercial sources (Figure 3.16). This pDNA expression vector family comprises a T7 RNA polymerase promoter for transcription, hence... 

Methods in which the amount of mRNA has been amplified to produce labeled cRNA, by incorporating a T7 RNA polymerase promoter site into one end of the cDNA followed by in vitro transcription are also widely used [51]. In this method, quantitative estimates of the amount of each transcript in a given sample can be calculated. In single-sample labeling experiments, a reference RNA may be spiked in during sample labeling and hybridization to facilitate quality control of the process as well as for comparison of data between different arrays. 

Radiolabelled (a32p) CTP is incorporated into multiple RNA copies using a commercial kit transcribing from a T7 RNA polymerase promoter. ... 

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