Galactose operon and Histidine operon

Gene expression in prokaryotes .

1. BANGALORE UNIVERSITY JB CAMPUS BANGALORE DEPARTMENT OF MICROBIOLOGY, BIOTECHNOLOGY & FOOD TECHNOLOGY TOPIC : GALACTOSE & HISTIDINE OPERON GUIDED BY : Dr. RAVIKIRAN.T PRESENTED BY : PUNITH KUMAR.S DEPT.OF BIOTECHNOLOGY M.Sc II Sem. BIOTECHNOLOGY BANGALORE UNIVERSITY

2. Contents : Operon Galactose operon Structural organization Metabolism Regulation Histidine operon Structural organization Regulation Conclusion Reference

3. OPERON An operon is a group of closely linked genes (structural and regulatory gene),which regulate the metabolic pathway in prokaryotes ,but not in eukaryotes. In eukaryotes, each gene is made up of an individual mRNA and each gene has its own promoter . A bacterium contains thousands of gene, when all genes functions at a same time the use will be flooded with enzymes and proteins. So, the genes for the required enzyme at that particular time are switched on and the other genes are switched off. This on and off mechanism is explained by the operon model. HISTORY : Operon were first identified as mode of expression control in 1961by Francois Jacob and Jacques Monod .

4. Galactose operon Gal operon is a prokaryotic operon. Codes enzymes for an amphibolic pathway of D-galactose metabolism . Gal operon performs catabolic and Anabolic function Catabolic Function : Galactose is used as Carbon source Catabolic Function : UDP Galactose UDP glucose Anabolic Function : UDP glucose UDP galactose This leads to the formation of lipopolysaccharide (cell wall component)

5. Structural organization of operon

6. Structure Operator 2 O E = Operator external O I = Operator internal Promoter 2 Overlapping P1 & P2 = Transcription start site hbs = histone unit binding site Activator sequence (AS) Structural genes 4 Gal E = UDP – galactose – 4 – epimerase Gal T = galactose – 1 – phosphate uridylyl transferase Gal K = galactokinase Gal M = mutarotase

7. This operon when performs catabolism. Galactose imported into the cell by permease Or galactose formed intercellularly by disaccharide metabolism In galactose metabolism , β – D galactose is not utilized , thus its converted into α -D galactose.

8. Leloir pathway of D-Galactose metabolism

9. REGULATION OF GAL OPERON 1) Gal R mediated DNA loop formation / repression Gal R gene encode tetrameric repressor that binds to 2 operators one located internally and one located externally This process requires presence of histone like protein (HU) and supercoiled DNA with operators in same face. Interaction of two operator bound Gal R results in formation of DNA loop (represosome) Looping of DNA blocks the access of RNA polymerase to promoters or inhibits formation of open complex. Structural genes are not transcribed .

10. 2) Gal R binds a dimer to O E A) Inhibits Promotor P 1 ( transcription off ) Inhibits formation of open promotor complex through RNA polymerase contact with α CTD Also causes CONTACT INHIBITION OF P 1 .

11. B) Activates promoter P 2 ( transcription on ) Allows low levels structural genes to be expressed NOTE: The Gal R – O I complex will not cause any repression because of presence of ANTI-PAUSE sequence in intermediate upstream area.

12. 3) Induction of Gal Operon by D - galactose Condition – 1 Increased level of D – galactose ( both α & β ) in medium Bind to allosteric site of repressor ( dimer ) Resulting in conformational change of protein repressor Suppress interaction of repressor with RNA P and DNA Induce operon and increase rate of galactose metabolism.

13. Condition – 2 : DNA looping D-galactose first breaks Gal R – Gal R tetramers into , Individual Gal R – O E and Gal R – OI dimer ( P 1 is repressed and P 2 is activated ) Gal R – O I ( dissociated by D – Galactose ) Gal R – O E ( dissociated and P 1 is activated )

14. 4) Regulation of CRP-CAMP complex ( CCC) CRP-cAMP complex binds at activation site (AS) Blocks RNA polymerase to form open complex with P 2 ( Transcription stops ) CRP-cAMP leads to formation of closed complex at P 1 with RNA polymerase that further isomerize to open complex ( Transcription ON ) NOTE : When glucose concentration is high in medium CRP-cAMP concentration will be low Basal level transcription occurs at P 2 by formation of binary complex cAMP and its receptor protein CRP complex enhances P 1 but represses P 2

15. HISTIDINE OPERON Histidine operon and its regulation of action in the bacterium Salmonella typhimurium . The bacteria controls its rate of histidine biosynthesis by 2 ways : 1. Intracellular concentration rises and feedback inhibition shuts down the pathway When histidine is being transported into the cell, the intracellular concentration rises and feedback inhibition shuts down the pathway. When external histidine is exhausted , the histidine pool fails until feedback inhibition is retrieved and biosynthesis of the amino acid is resumed .

16. 2. Repression control, Governs the intracellular concentration of biosynthetic enzymes. This method is considerably slower in action than feedback inhibition although the enzymes concentration may begin new steady state level is reached. Presence or absence of external histidine is not the only factor regulating the rate of histidine biosynthesis. The bacterium also keeps its rate of synthesis of amino acid in line with its growth rate.

17. Structural organization of operon Histidine operon contains cluster of 9 structural gene his G,D,C,B,H,A,F,I,E which codes for enzymes aids in the pathway of synthesis of histidine from 5-phosphoribosyl 1-pyrophosphate(PRPP). There are 5 regulatory genes viz. his R,U,S,T and W that are associated with the operon but not closely linked to it . The gene his R codes for histidine tRNA , while his S codes for histidyl-tRNA synthetase. The histidine operon lacks an operator region and a CRP site.

18. Because of this lack of an operator region , the transcriptional control in histidine operon solely takes by attenuation. The transcription produces a single polycistronic mRNA that is about 7300 nucleotides long , extending from primary promoter to Rho-independent terminator. 2 week internal promoters , Hisp2 and Hisp3 are located within the HisC and HisF genes respectively.

19. Regulation : Control of Transcription Initiation & Elongation Transcription of the his operon is about four fold more efficient in bacteria growing in minimal glucose medium than when growing in rich medium. This form of control is called metabolic regulation , adjusts the expression of the operon to the amino acid supply in the cell. It is mediated by “alarmone” (ppGdd) guanosine 5’-diphosphate 3’-diphosphate , which is the effector of the stringent response . The alarmone regulates the his operon positively by stimulating the primary promoter hisp1 under conditions of moderate amino acid starvation.

20. Regulation : Attenuation control of the histidine operon Attenuation is the modification of gene expression by events that influence aspects of transcription other than initiation of transcription . The attenuator is located in the leader region of the RNA transcript that lies between the promoter and the 1 st structural gene. It comprises a segment coding for the leader peptide followed by a terminator sequence .

21. The his-specific regulatory element is transcribed in a 180-nucleotide RNA leader , which exhibits two prominent features: ( i ) A 16-residue coding sequence in the leader peptide –coding region , including seven consecutive codons specifying histidine . The amino acid sequence is : (ii) overlapping regions of dyad symmetry capable of folding into mutually exclusive, alternative secondary structure that signal either transcription termination or anti termination. There are 3 stems in the termination configuration and 2 in the antitermination configuration.

22. Negative regulation : RNA Termination Translation control of His operon, transcription is determined by ribosomes occupancy of leader RNA which in turn depends given the peculiar composition of His leader peptide on the availability of His tRNA . When histidine is present in sufficient amount , the supply of histidyl tRNA is plentiful. This permits the translating ribosome to pass across the leader sequence of RNA without pausing. The relevant codons and adjacent nucleotides are free to base pair , resulting in the formation of the RNA terminator this prevents the translation of his operon structural genes .

23. Negative regulation : RNA Termination In case of severe limitation of intracellular pool of all charged tRNAs, translation of leader peptide fails to initiate: under these condition, the A:B, C:D, E:F stem loop structures from sequentially, producing a strong transcription termination.

24. Positive regulation : Anti-termination When histidine is in low concentration , the supply of histidyl tRNA is limited, this result in the ribosome pausing or stalling as it passes across the leader sequence and masking the codons, this permits the base downstream to base pair and from the antitermination structure , which in turn prevents the formation of the terminator. Translation of the his operon structural genes therefore takes place.

25. Conclusion The operon , genetic regulatory system found in prokaryotes ,in which genes coding for functionally related proteins are clustered along the DNA . This feature allows controlled protein synthesis according to the needs of cell. The gal operon of E.coli plays a major role in cellular metabolism by encoding enzymes that catalyze conversion of D-galactose to energy sources as well as to anabolic substrates. The histidine operon have served as powerful model system for studying fundamental evolutionary , metabolic ,physiological and genetic processes , such as gene duplication , transposition , mutagenesis, including the widely used Ames test.

26. Reference Books John S. Kovach. Robert F. Goldberger. Journal of bacteriology. 2006. volume 97. page no : 1283-1290. Chang GW, Roth jr, Ames BN (2009). Histidine regulation in salmonella typhimurium. 8.mutation of his T gene. J Bacteriol 108:410-414 Journal Irani M.H, Orosz L, Adhya S(1983). A control element within a structural gene; The gal operon of Escherichia coli.Cell 32(3):783-788.https://doi.org/10.1016/0092-8674(83)90064-8. Dale E A Lewis , Sankar Adhya (2015). Molecular mechanism of transcription initiation at gal promoters and their multi-level regulation by GalR , CRP and DNA loop.Biomolecules.5(4):2782-807.https://doi.org/10.3390/biom5042782.

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