Part:BBa_K842003
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:
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.
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 *)
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3
Illegal PstI site found at 91 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3
Illegal PstI site found at 91 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3
Illegal PstI site found at 91 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3
Illegal PstI site found at 91 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 426
Illegal SapI.rc site found at 451
Illegal SapI.rc site found at 607
| None |