control of gene expression by sigma factor and post transcriptional control

explanation of control of gene expression by sigma factor and decription of sigma factor and detailed explation of post transcriptional control by antisense technology and rna i

1. CONTROL OF GENE EXPRESSION BY SIGMA FACTOR

2. Introduction Sigma factors are subunits of all bacterial  RNA polymerases  that are responsible for determining the specificity of promoter  DNA binding  and efficient initiation of  RNA synthesis  (transcription). The first sigma factor discovered was the sigma70 (σ 70 ) of the highly studied bacterium  Escherichia coli . Its discovery in 1968 was an unexpected result of trying to understand the  subunit structure  of the RNA  polymerase . It was found that the RNA polymerase exists in two forms. A core polymerase (with subunit structure α 2 ββ′ω) can transcribe DNA into RNA inefficiently and nonspecifically . When the sigma subunit, σ 70 , is added, it can bind to core and form a holoenzyme (α 2 ββ′σω 70 ) that is capable of specific binding at the beginning of genes (promoters) and efficient initiation of transcription.

3. It was hypothesized that multiple sigma factors would be found in  E. coli , each capable of directing the core polymerase to transcribe a class of genes. In this way, by regulating the abundance of each active sigma factor, the cell could co-ordinately regulate groups of genes with common functions. During the past 40 years, multiple sigma factors have indeed been found. The seven sigma factors of  E. coli  are listed in Table 1 along with their gene names, molecular weights, consensus promoter DNA binding sites, and classes of genes that they regulate. The type of sigma factor that is used in this process varies and depends on the gene and on the cellular environment. The sigma factors identified to date are characterized based on molecular weight and have shown diversity between bacterial species as well. Once the role of the sigma factor is completed, the protein leaves the complex and RNA polymerase will continue with transcription

4. The regulation of sigma factor activity is critical and necessary to ensure proper initiation of transcription. The activity of sigma factors within a cell is controlled in numerous ways. Sigma factor synthesis is controlled at the levels of both transcription and translation. Often times, sigma factor expression or activity is dependent on specific growth phase transitions of the organism. If transcription of genes involved in growth is necessary, the sigma factors will be translated to allow for transcription initiation to occur. However, if transcription of genes is not required, sigma factors will not be active. In specific instances when transcriptional activity needs to be inhibited, there are anti-sigma factors which perform this function. The anti-sigma factors will bind to the RNA polymerase and prevent its binding to sigma factors present at the promoter site. The anti-sigma factors are responsible for regulating inhibition of transcriptional activity in organisms that require sigma factor for proper transcription initiation

5. Antisigma Factors Inactivate Sigma Sigma factors may be inhibited by proteins known as  antisigma factors . These 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. σF is kept inactive by an antisigma factor ( SpoIIAB ). This antisigma factor is, in turn, displaced from σF by an  anti-anti-sigma factor  ( SpoIIAA ). This event triggers the cascade of  gene activation  described earlier.

6. The regulation of alternative sigma factors by binding to antisigma factors is widespread. However, their release by anti-anti-sigma factors is fairly rare.  Two other major mechanisms exist for releasing sigma factors from antisigma factors—regulated proteolysis, and direct sensing.  regulated proteolysis  the antisigma factor is degraded by a  protease  that responds to some incoming signal. direct sensing  the incoming signal affects the antisigma factor directly. For example, some antisigma factors respond to  oxidative stress  by changing shape and then releasing the sigma factor.  A clinically important example of regulated proteolysis is the production of mucus by  Pseudomonas aeruginosa . This bacterium often infects the lungs of  cystic fibrosis  patients, where it switches on the genes for mucus production. The bacterial mucus clogs the patient’s airways and is a major contributor to the symptoms of the disease. 

7. T he material made by  Pseudomonas  is  alginate , an acidic  polysaccharide  that is a repeating polymer of mannuronic and  glucuronic acids . This is chemically distinct from the true mucus made by animal cells, which consists of  glycoproteins  with many short side chains of  galactose , N-acetyl-galactosamine, and  N-acetyl-neuraminic acid .  Alginate synthesis is under control of the alternative sigma factor AlgU and the antisigma factor MucA . The protease AlgW resides in the  periplasmic space  and responds to signals from the environment. AlgW then cleaves MucA , which is a  trans-membrane protein , so liberating AlgU and activating mucus production . Stationary Phase σ Factors Sigma factors include numerous types of factors. The most commonly studied sigma factors are often referred to as a RpoS proteins as the rpoS genes encode for sigma proteins of various sizes. In  E. coli , the RpoS is the regulator of growth phase genes, specifically in the stationary phase. The RpoS is critical in the general stress responses and can either function in promoting survival during environmental stresses, but can also prepare the cell for stresses. Specifically, the translational control of the sigma factor is a major level of control.

8. The translational control of sigma factors It involves the presence and function of small noncoding RNAs . Using RpoS proteins as the focus, the RpoS expression and transcription is regulated at the translational level. Small noncoding RNAs are able to sense environmental changes and stresses resulting in increased expression of RpoS protein. The small noncoding RNAs are able to specifically increase the amount of rpoS mRNA that undergoes translation. The resultant increase of RpoS protein is based on the cellular environment and its needs. There are numerous classes of small noncoding RNAs that function in RpoS regulation, including DsrA , RprA and OxyS . These small noncoding RNAs are capable of sensing changes in temperature ( DsrA ), cell surface stress ( RprA ) and oxidative stress ( OxyS ). These RNAs can induce activation of rpoS translation. However, there are small noncoding RNAs, such as LeuO , that are capable of inhibiting rpoS translation as well via repression mechanisms. The regulation of rpoS translation is complex and involves cross- signaling and networking of numerous proteins and the regulatory small noncoding RNAs.

9. Alternative sigma factors   alternative sigma factors provide an additional strategy to regulate  gene transcription  activity by altering RNA polymerase affinity for promoter sequences and thereby inducing major changes in global transcription patterns. Alternative sigma factors are distributed into two main families: the σ 70 -family, a group that exhibits high  structural homology  to housekeeping sigma factors and the unrelated σ 54 -family, which relies on an additional ATP-dependent transcription factor to initiate transcription at the promoter The σ 70 -family of sigma factors has been divided into four groups based on protein domain structure and amino acid sequence conservation Group I sigma factors consist of the housekeeping sigma factors, which, when tested, are known to be essential for viability.  Group II and III alternative sigma factors are involved in various cellular processes, such as development, general stress response, or virulence. Sigma factors in Group IV were only recognized some 20 years ago, but they are now known to be the largest and most diverse group of sigma factors with more than 40 subgroups identified by  phylogenetic  analysis of sequenced  bacterial genomes Group IV sigma factors appear to have limited and specific functions, which often relate to extracytoplasmic stresses (Group IV sigma factors are also referred to as extracytoplasmic factors). Group IV sigma factors are important components of bacterial   signal transduction   networks .

10. Post transcriptional control

11. Introduction Even after a gene has been transcribed, gene expression can still be regulated at various stages. In eukaryotic cells often controlled primarily at the level of transcription. However, that doesn't mean transcription is the last chance for regulation. Later stages of gene expression can also be regulated. RNA is transcribed, but must be processed into a mature form before translation can begin. This processing after an RNA molecule has been transcribed, but before it is translated into a protein, is called post-transcriptional modification.  post-transcriptional step can also be regulated to control gene expression in the cell. If the RNA is not processed, shuttled, or translated, then no protein will be synthesized. RNA splicing, the first stage of post-transcriptional regulation In eukaryotic cells, the RNA transcript often contains regions, called introns, that are removed prior to translation. The regions of RNA that code for protein are called  exons . After an RNA molecule has been transcribed, but prior to its departure from the nucleus to be translated, the RNA is processed and the introns are removed by splicing. Splicing is done by spliceosomes, ribonucleoprotein complexes that can recognize the two ends of the intron, cut the transcript at those two points, and bring the exons together for ligation.

12. This alternative splicing can be haphazard, but more often it is controlled and acts as a mechanism of gene regulation, with the frequency of different splicing alternatives controlled by the cell as a way to control the production of different protein products in different cells or at different stages of development. Control of RNA Stability Before the mRNA leaves the nucleus, it is given two protective “caps” that prevent the ends of the strand from degrading during its journey. 5′ and 3′ exonucleases can degrade unprotected RNAs. The  5′ cap , which is placed on the 5′ end of the mRNA, is usually composed of a methylated guanosine triphosphate molecule (GTP). The  poly-A tail , which is attached to the 3′ end, is usually composed of a long chain of adenine nucleotides. These changes protect the two ends of the RNA from exonuclease attack. Once the RNA is transported to the cytoplasm, the length of time that the RNA resides there can be controlled. Each RNA molecule has a defined lifespan and decays at a specific rate. This rate of decay can influence how much protein is in the cell. If the decay rate is increased  the RNA will not exist in the cytoplasm as long, shortening the time available for translation of the mRNA to occur. if the rate of decay is decreased  the mRNA molecule will reside in the cytoplasm longer and more protein can be translated. This rate of decay is referred to as the RNA stability

13. RNA-binding proteins Binding of proteins to the RNA can also influence its stability. Proteins called  RNA-binding proteins , or RBPs, can bind to the regions of the mRNA just upstream or downstream of the protein-coding region. These regions in the RNA that are not translated into protein are called the  untranslated regions , or UTRs. They are not introns (those have been removed in the nucleus). Rather, these are regions that regulate mRNA localization, stability, and protein translation. The region just before the protein-coding region is called the  5′ UTR , whereas the region after the coding region is called the  3′ UTR  . The binding of RBPs to these regions can increase or decrease the stability of an RNA molecule, depending on the specific RBP that binds.

14. Anti sense technology In this step, antisense oligonucleotides inhibit post-transcriptional modification or RNA splicing. Once the mRNA is transcribed from DNA, it undergoes several modification including 5’ capping, polyA tail and intron removal. After post transcriptional modification, mRNA is transported out from nucleus to cytoplasm. Once the mRNA is hybridized with antisense oligonucleotide, the double stranded RNA duplex cannot be transported out to cytoplasm. Removal of intron is essential for RNA splicing because intron are non-coding sequences. In this process, the oligonucleotide-based antisense agent is used which binds with specific sequence of pre-mRNA preventing intron-excision.

15. Small regulatory RNAs A recently discovered class of regulators, called  small regulatory RNAs , can control mRNA lifespan and translation These include under RNA interference(RNA i ) it is done by different RNA’s such as miRNA. shRNA . siRNA. RNA interference  ( RNAi ) is a biological process in which  RNA  molecules  inhibit gene expression  or translation, by neutralizing targeted  mRNA  molecules We need RNAi :- 1. miRNA:- ssRNA 2. siRNA :- dsRNA 2. RISC :- RNA induced silencing complex, that cleaves mRNA. RNA-induced silencing complex , or  RISC , is a multiprotein  complex  that incorporates one strand of a small interfering RNA (siRNA) or micro RNA (miRNA).  RISC  uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it finds a complementary strand, it activates RNase and cleaves the RNA

16. The RISC-loading complex (RLC) is the essential structure required to load dsRNA fragments into RISC in order to target mRNA. The RLC consists of dicer, the transactivating response RNA-binding protein ( TRBP ) and Argonaute 2. Dicer  is an RNase III  endonuclease  which generates the dsRNA fragments to be loaded that direct RNAi. TRBP  is a protein RNA-binding  domains . Argonaute 2  is an RNase and is the catalytic centre of RISC ENZYMES:- 1. DICER:- Produces 20-21nt cleavages that initiate RNAi. 2. DROSHA:- Cleaves base hairpin in to form pre miRNA; Which is later processed by Dicer

17. miRNA It is a non coding RNA molecule of approx 21-23 nt. that inhibits the mRNA expression is known as miRNA. miRNAs come from hairpin precursors generated by the RNaseIII enzymes  Drosha  and  Dicer The formation of micro RNA (miRNA) consists of three important steps:- Formation of primary miRNA. Formation of precursor miRNA. Formation of mature functional miRNA.

18. siRNA A lso as short interfering RNA or silencing RNA. It is a class of double - stranded RNA molecules, similar to  miRNA , and operating within the  RNA interference  (RNAi) pathway Naturally occurring siRNAs have a well-defined structure that is a short (usually 20 to 24- bp )  double-stranded RNA  (dsRNA) with  phosphorylated  5' ends and  hydroxylated  3' ends with two overhanging nucleotides. The  Dicer   enzyme  catalyzes production of siRNAs from long  dsRNAs  and  small hairpin RNAs It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, resulting no translation siRNAs can also be introduced into cells by  transfection . siRNAs come from long dsRNA precursors derived from a variety of single-stranded RNA ( ssRNA ) precursors, such as sense and antisense RNAs. siRNAs also come from hairpin RNAs derived from transcription of inverted repeat regions. siRNAs may also arise enzymatically from non-coding RNA precursors

19. Mechanism The mechanism by which natural siRNA causes gene silencing through repression of translation occurs as follows Long dsRNA (which can come from hairpin, complementary RNAs, and RNA-dependent RNA polymerases) is cleaved by an endo-ribonuclease called  Dicer . Dicer cuts the long dsRNA to form short interfering RNA or siRNA; this is what enables the molecules to form the RNA-Induced Silencing Complex (RISC). Once siRNA enters the cell it gets incorporated into other proteins to form the  RISC . Once the siRNA is part of the RISC complex, the siRNA is unwound to form single stranded siRNA. The strand that is thermodynamically less stable due to its base pairing at the 5´end is chosen to remain part of the RISC-complex The single stranded siRNA which is part of the RISC complex now can scan and find a complementary mRNA Once the single stranded siRNA (part of the RISC complex) binds to its target mRNA, it induces  mRNA  cleavage. The mRNA is now cut and recognized as abnormal by the cell. This causes degradation of the mRNA and in turn no translation of the mRNA into amino acids and then proteins. Thus silencing the gene that encodes that mRNA.

20. Short hairpin RNA A  short hairpin RNA  or  small hairpin RNA  ( shRNA /Hairpin Vector) is an artificial  RNA  molecule with a tight hairpin turn that can be used to silence target gene expression via  RNA interference  (RNAi). Expression  of shRNA in cells is typically accomplished by delivery of  plasmids  or through viral or bacterial  vectors . In the creation of sh RNA first,  polymerase III  promoters such as U6 and H1 were used; however, these promoters lack spatial and temporal control.  As such, there has been a shift to using  polymerase II  promoters to regulate shRNA expression. Delivery Expression of shRNA in cells can be obtained by delivery of  plasmids  or through viral or bacterial  vectors . Delivery of plasmids to cells through transfection to obtain shRNA expression can be accomplished using commercially available reagents  in vitro . However, this method is not applicable  in vivo  and thus has limited utility  examples- adeno-associated viruses  (AAVs),  adenoviruses , and  lentiviruses , where as bacterial vectors such as e.coli is a recent approach

21. Mechanism of action Once the vector has integrated into the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase III depending on the promoter choice. The product mimics pri -microRNA ( pri -miRNA) and is processed by  Drosha . The resulting pre-shRNA is exported from the nucleus by Exportin 5. This product is then processed by  Dicer  and loaded into the  RNA-induced silencing complex  (RISC). The sense (passenger) strand is degraded. The antisense (guide) strand directs RISC to mRNA that has a complementary sequence. In the case of perfect complementarity  RISC cleaves the mRNA. In the case of imperfect complementarity  RISC represses translation of the mRNA. In both of these cases, the shRNA leads to target gene silencing. Antisense  therapy means the selective, sequence-specific inhibition of gene expression by single-stranded DNA oligonucleotides. In contrast,  RNA interference  ( RNAi ) is triggered by double-stranded  RNA  (dsRNA) and causes sequence-specific mRNA degradation of single-stranded target  RNAs  in response to dsRNA.