Difference between revisions of "Part:BBa K842003"

Latest revision as of 14:15, 27 October 2017

fliA

fliA is an alternate sigma factor for the class 3 flagella operons. When transcribed, it forms a component of RNA polymerase sigma 28.

Used for reconstitution of flagella apparatus in E. coli B strains.

32 and 28 belong to the 3rd group of Sigma factors. Sigma factors of that group contain of 2, 3 and 4 areas and structurally are really diverse. 28 is involved in the synthesis of flagell in E.coli(Paget, 2015). For that work of sigma factor the great value has a weaker capacity to the promoter melting. It has been shown that having the strict consensus in the promoter region is important for 28 , it provides a strict specifity of recognition in the initiation of transcription(Koo et al, 2009).

Shown consensus:

(doi:10.1093/nar/gkm456)

It may be explained with that fact, that this type of sigma factors may not have one or several amino acid residues, required for villiany -11A when you initiate promoter region melting(Feklístov et al, 2014).

Pic. 1. Alignment of amino acid sequences of the alternative Sigma relative to the main one. Area 3. 2 is shown in red.

(doi:10.1093/nar/gkt1384)

When performing transcription involving this sigma sub-unit it is necessary to consider the factors that may influence the product output (that is RNA transcript in our case). The main factors influencing the function of the enzyme are salt concentration in the reaction buffer and the temperature of the experiment. Standard conditions for transcription are considered to be 40-50 mM NaCl buffer. However, other salts and concentrations have been tested and it has been discovered that when using KCl instead of NaCl the reaction optimum is not offset and remains

at the concentration rate of 40-50 mM. But when using sodium glutamate and sodium acetate instead of sodium chloride the reaction optimum shifts to high concentration regions (300-400 mM). Besides, the influence of temperature on reaction efficiency should be considered. Here it should be noted that the function optimum of RNA polymerase containing an s70 sub-unit is observed at 37°C while for an enzyme containing an s38 sub-unit this value goes down to 30°C (for achieving maximum transcription reaction efficiency). (Promoter Selectivity of Escherichia coli RNA Polymerase sF Holoenzyme Involved in Transcription of Flagellar and Chemotaxis Genes TAPAS KUMAR KUNDU,1 SHUICHI KUSANO,1,2 AND AKIRA ISHIHAMA1 *)

As sigma subunits account for promoter binding specificity, for conducting experiments with RNAP it is important to understand different promoters’ strength. Therefore, we decided to examine and compare the activity of E. coli RNA polymerase with two different promoters characterized for this sigma - flgMp and modAp1. It was discovered that promoter complex stability for modAp1 was higher than for flgMp. The half-life of flgMp and modAp1 promoters was 38s and 1800s, accordingly. Standard deviations were plotted based on the results of three experiments.

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Besides, it was demonstrated that the efficiency of synthesis differed for two promoters as well: the efficiency of synthesis for flgMp was higher than that one for modAp1.

Experiment design: Promoter sequences were taken from RegulonDB and amplified using PCR with genomic DNA of E. coli.

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 Highlights: -35 (yellow), -10 (green) and +1 base (purple).

To measure the kinetics of promoter complex dissociations, competitive inhibition of transcription by heparin was used. Full-length products were synthesized in the following conditions: RNAP core enzyme – 200 nmol, σ-subunit – 500 nmol. Heparin was being added to the reaction mixture containing an open RNAP promoter complex with a correspondent promoter until its concentration reached 10mcg/ml. At different intervals after that (20′′, 40", 1′, 3′, 10′, 30′) samples were taken from the mixture and transcription was initiated in them by adding a mixture of nucleotides: ATP, CTP and GTP in equal concentrations (200 µmol), 10 µmol of UTP, and 2.5 µCi of α-[32-P]-UTP (3000 Ci/mmol). Each sample was incubated for 3 minutes at 37°С (in BIOSAN WB-4MS water thermostat); after that the reaction was terminated by adding equal amounts of stop-buffer. In the beginning of the experiment a ‘null’ sample had been taken from the reaction mixture (control without heparin) to which the same mixture of nucleotides was added. The results were visualized using a phosphorimager; the quantitative analysis of the results was carried out with the help of ImageQuant.

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Cloning and Uses: This part was generated via PCR from the E. coli strain DH5α and cloned into the vector pSB1C3. It is to be used in conjunction with the entire flagella synthesis genetic pathway to reproduce a complete flagella apparatus.







Sequence and Features