role of sigma factor.pptx

sigm factors in e coli in transcription

1. Role of Sigma factors in Regulation ( Escherichia coli  ( E .  coli ) AGM-602 –Microbial Physiology and Regulation BY Namadara Sandhya 1 st year PhD 202211004 Department of Agriculture Microbiology

2. What is Sigma factor  (σ factor or specificity factor )? Sigma factors are multi-domain subunits of bacterial RNA polymerase (RNAP) that play critical roles in transcription initiation, including the recognition and opening of promoter elements to form an initial “closed” complex ( RPc ), stabilisation of the “open” complex ( RPo ) in which DNA around the transcription start site is melted, interaction with transcription activators, the stimulation of the early steps in RNA synthesis ( Saecker et al .,2011) The sigma factor, together with RNA polymerase core enzyme  consists of five subunits including  α (two copies), β, β' and ω subunits , is known as the RNA polymerase  holoenzyme . ( Paget, Mark S . ,2015)

3. These five subunits form the RNAP core enzyme  responsible for RNA synthesis using DNA as template and ribonucleotide ( rNTP ) as substrate . Every molecule of RNA polymerase holoenzyme contains exactly one sigma factor subunit, which in the model bacterium  Escherichia coli  is one. Because of the absence of the sigma factor, E.coli RNA polymerase core enzyme is unable to recognize any specific bacterial or phage DNA promoters. Instead it transcribes RNA from nonspecific initiation sequences.  Addition of sigma factors will allow the enzyme to initiate RNA synthesis from specific bacterial and phage promoters. ( Paget, Mark S . ,2015)

4. Structure of Sigma factor Sigma σ Factors play 3 major roles in the RNA synthesis initiation process: they ( i ) target RNAP holoenzyme to specific promoters, (ii) melt a region of double-stranded promoter DNA and stabilize it as a single-stranded open complex, and (iii) interact with other DNA-binding transcription factors to contribute complexity to gene expression regulation schemes. σ Factors are encoded by genes rpo .those protein molecules ranges from 17-80 K.D The number of sigma factors varies between bacterial species. In E. coli  has seven sigma factors. Sigma factors are distinguished by their characteristic molecular weights. For example, σ 70  is the sigma factor with a molecular weight of 70  kDa .

5. In E.coil always sigma 70 is present ,required for normal growth of ecoil Additional experiments demonstrated that σ70 was catalytic: Once initiation occurred, σ70 could dissociate from one RNAP molecule Thus, RNAP has two distinct forms: holoenzyme ( α2ββω+σ), for initiation, and core ( α2ββω), for elongation Anti-sigma factors are responsible for inhibiting sigma factor function thus, inhibiting transcription.

6. Dissociable σ factors, which bind core RNAP to form holoenzyme , direct key aspects of the initiation process, including recognition of promoter DNA and melting of the DNA to expose the transcription start site. This process was originally described as a ‘‘ σ cycle’’ (Figure 1), in which σ associates with RNAP to orchestrate initiation and then dissociates after the transition to a stable elongation complex (EC) is complete ( Travers and Burgess, 1969; Chamberlin, 1976). Once RNAP finishes transcription and releases DNA and RNA, it is free to be bound anew by σ and begin another cycle of transcription. The key feature of the σ cycle is the ability of RNAP to be reprogrammed rapidly by different σ in each new round of transcription. ‘‘ σ cycle’’

7. They can be classified into two distinct families based on their homology to two factors in Escherichia coli: the primary factor 70 that is responsible for the bulk of transcription during growth; and the structurally unrelated 54 (or N) that directs transcription in response to environmental signals, and requires the input of enhancer proteins and ATP hydrolysis to drive DNA melting The σ proteins are composed of a variable number of structure domains connected by flexible linkers. The simplest σs have two domains (Group 4 or ECF σs : σ2,σ4), some have three domains (Group 3 σs : σ2, σ3, σ4), and the housekeeping σs have four domains(σ1.1, σ2, σ3, σ4). Except for σ1.1, each domain has DNA-binding determinants: σ4,-35 motif; σ3, extended -10 motif; σ2, -10 and discriminator motifs.

8. Heat-Shock Response in Escherichia coli Cells respond to a sudden increase in temperature by increasing their rate of synthesis of a small number of proteins and it is called the heat-shock response the proteins synthesized in response to heat stress are called the heat-shock proteins (HSPs). Heat shock responses are maintained by σ32/ and σ24 stress condition /high temp causes unfolding of protein (they will be having particular pattern ,if it is distrubed it will be deactivated )

9. E. coli, like other organisms, responds to heat shock by rapidly up-regulating several proteins, including chaperones. The heat shock sigma factor, sigma 32 (σ32), a transcription factor, plays a pivotal role in this response. The level of σ32 is normally kept low through a DnaK /J mediated degradation. Elevated temperature rapidly increases the σ32 level and initiates a heat-shock response. The increased level of σ32 leads to the synthesis of large numbers of molecular chaperones and proteases, that in turn act as a negative feedback on the level of σ32. Chaperones refold proteins efficiently and rapidly. A posible way for the up-regulation of free σ32 levels would be to destabilize the σ32:DnaK:DnaJ complex initiated via a conformational change in σ32 structure at elevated temperatures.

10. Anti-sigma factors In bacteria, the regulation of gene expression is the basis for adaptability, morphogenesis, and cellular differentiation. From all the different regulatory layers, regulation of transcription initiation is a very important step for controlling gene expression. Each sigma factor has an associated anti-sigma factor which regulates it. These anti-sigma factors are divided into either cytoplasm or inner membrane bound anti-sigma factors.   Cytoplasmic bound anti-sigma factors are made up of FlgM , DnaK , RssB , & HscC .  Inner membrane bound anti-sigma factors are made up of FecR & RseA .  Anti-sigma factors are simultaneously transcribed with their associated sigma factor.

11. In prokaryotes,  E. coli  has seven different sigma factors depends on the environment condition. Each one specific anti-sigma factors (Trevino et al., 2013)

12. The mechanism for releasing cytoplasmically -located σ factors in response to signals that often stem from the external environment . They can be broadly divided into partner-switching, direct sensing and regulated proteolysis mechanisms . In the case of partner-switching and regulated proteolysis, an emerging theme is the integration of distinct signals involving separate input pathways that enable σ activation in response to varied environmental and physiological cues.

13. Anti-sigma factors bind to specific sigma factors and prevent them from associating with RNA polymerase  . When σF is first made in the developing spore, it is inactive. Unlike σE and σK , which need to be activated by the proteolysis of an inactive precursor protein, σF is kept inactive by an anti-sigma factor ( SpoIIAB ). This anti-sigma factor is, in turn, displaced from σF by an  anti-anti-sigma factor  ( SpoIIAA ). This event triggers the cascade of gene activation described above.

14. Figure 1.  Anti-Sigma Factor The anti-sigma factor SpoIIAB binds to σF and inactivates it. When the cell receives an external signal, the phosphorylated form of SpoIIAA , an anti-anti-sigma factor, loses its phosphate and engages SpoIIAB . This releases σF , which is then free to activate the sporulation cascade shown above in Figure

15. References Paget, Mark S. 2015. "Bacterial Sigma Factors and Anti-Sigma Factors: Structure, Function and Distribution"  Biomolecules  5, no. 3: 1245-1265. https://doi.org/10.3390/biom5031245 Saecker , R.M.; Record, M.T.; Dehaseth , P.L. Mechanism of bacterial transcription initiation: RNA polymerase—Promoter binding, isomerization to initiation-competent open complexes, and initiation of RNA synthesis. J. Mol. Biol. 2011, 412, 754–771.] Maria C. Davis, Christopher A. Kesthely , Emily A. Franklin, and Shawn R. MacLellan . The essential activities of the bacterial sigma factor.  Canadian Journal of Microbiology .  63 (2): 89-99. https://doi.org/10.1139/cjm-2016-0576 Burgess RR, Travers AA, Dunn JJ, Bautz EK. Factor stimulating transcription by RNA polymerase. Nature. 1969 Jan 4;221(5175):43-6. doi : 10.1038/221043a0. PMID: 4882047. Treviño -Quintanilla LG, Freyre-González JA, Martínez -Flores I (September 2013).  " Anti-Sigma Factors in E. coli: Common Regulatory Mechanisms Controlling Sigma Factors Availability".  Current Genomics .  14  (6): 378–87. doi:10.2174/1389202911314060007. PMC 3861889. PMID 24396271