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Operons

Operons. Nilansu Das Dept. of Microbiology Surendranath College. Paper III Group A: Cellular and Molecular Biology Unit I 1. DNA Replication: (10) DNA-Replication-Meselson-Stahl experiment as evidence for semiconservative

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Operons

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  1. Operons Nilansu Das Dept. of Microbiology Surendranath College

  2. Paper III Group A: Cellular and Molecular Biology Unit I 1. DNA Replication: (10) DNA-Replication-Meselson-Stahl experiment as evidence for semiconservative replication; Mechanism of replication-Rolling-circle model & Theta (8) structure (bidirectional) 2. Transcription in prokaryotes: (15) Mechanisms (Initiation, elongation, termination); promoter structures, subunits of bacterial polymerases, functions and domains responsible for activity, elongationprocess, mechanism of termination, -dependent and independent termination; lac, trp, ara operons. 3. Mechanism of translation in prokaryotes: (15) Description of ribosomal cycle including phenomena of initiation, elongation, termination; description of factors involved in these processes; genetic code; tRNA: clover-leaf structure & function; rRNA: structure and function; role of aminoacyl tRNA synthetases. Non-ribosomal peptide synthesis: cyclic peptide antibiotics e.g. Gramicidin etc.

  3. Prokaryotic Gene Regulation Coordinate regulation of genes involved in similar functions

  4. Types of Control

  5. Operon • Unit of coordinate gene expression • Includes structural genes and their adjacent regulatory elements • Lac operon (inducible) • Ara operon (inducible) • Trp operon (repressible)

  6. Types of Operons

  7. Regulation of the Lac Operon crp O Pcrp Pi P Lac Z Lac Y Lac A I Structural Genes

  8. O Pcrp crp Pi P Lac Z Lac Y I Lac A Pol Transcription Z, Y, A mRNA Translation B-galactosidase Permease Acetylase RNA polymerase binds to the promoter and produces a polycistronic mRNA from the Lac Z, Y and A genes. All three proteins are produced. Transcription from the Lac Operon

  9. O Pcrp crp Pi P Lac Z Lac Y Lac A I Pol I mRNA crp mRNA Active repressor Inactive CAP protein Transcription from Pcrp and Pi is constitutive: always expressed in an unregulated fashion. Active repressor binds to operator and prevents RNA polymerase from reaching structural genes. Regulation of the Lac Operon: Low lactose, High glucose No mRNA produced No Z, Y, A proteins produced

  10. O Pcrp crp Pi P Lac Z Lac Y Lac A I Pol Transcription I mRNA crp mRNA Z, Y, A mRNA Translation + Lactose Active repressor Inactive CAP protein B-galactosidase Permease Acetylase Inactive repressor Lactose (inducer) binds to the repressor and inactivates it. RNA polymerase transcribes Lac Z, Y and A at low frequency. Regulation of the Lac Operon: High lactose, High glucose

  11. O Pcrp crp Pi P Lac Z Lac Y Lac A I Pol Transcription I mRNA crp mRNA Z, Y, A mRNA Translation + + Lactose Active repressor cAMP Inactive CAP protein B-galactosidase Permease Acetylase Active CAP protein Inactive repressor cAMP is produced when glucose levels are low. cAMP activates CAP. Active CAP binds to the promoter to increase RNA polymerase binding. RNA polymerase transcribes Lac Z, Y and A at HIGH frequency. Regulation of the Lac Operon: High lactose, Low glucose

  12. O Pcrp crp Pi P Lac Z Lac Y Lac A I Pol I mRNA crp mRNA + Active repressor cAMP Inactive CAP protein Active CAP protein Although RNA polymerase binding is enhanced by Active CAP, the operator is blocked by active repressor. RNA polymerase cannot transcribe Z, Y and A. Regulation of the Lac Operon: Low lactose, Low glucose No mRNA produced No Z, Y, A proteins produced

  13. CAP Protein Structure Allows Binding to DNA • Domains are regions on a protein with specific functions; motifs are characteristic structures within a domain • CAP has a DNA binding domain with a helix-turn-helix structural motif • Helices fit into the major groove on DNA

  14. Summary of Lac Operon Regulation Off Off On at low frequency On at high frequency

  15. Mutations of the Lac Operon O Pcrp Pi CRP P Lac Z Lac Y Lac A I Functional genes: I+ P+ O+ Z+ Y+ A+ The diffusible product of the I+ or IS allele can associate with an operator on the same piece of DNA (cis) or on a separate piece of DNA (trans).

  16. Mutations of the Lac Operon O Pcrp Pi CRP P Lac Z Lac Y Lac A I Functional genes: I+ P+ O+ Z+ Y+ A+

  17. Mutations of the Lac Operon O Pcrp Pi CRP P Lac Z Lac Y Lac A I Functional genes: I+ P+ O+ Z+ Y+ A+ A mutation in one structural gene does not affect the production of proteins from the other structural genes.

  18. B-Galactosidase Permease Lac Operon Mutations No lactose Lactose No lactose Lactose -- + -- + + + + + -- -- -- -- + + + + -- + -- + + + -- + -- + + + -- -- -- -- -- -- -- --

  19. Arabinose Operon

  20. C Protein Exerts Positive and Negative Control of the Ara Operon Arabinose present Arabinose absent

  21. Summary of Ara Operon Regulation OffC protein bound to O and I,Inhibiting transcription OffC protein bound to O and I On at low frequencyC protein + arabinose bound to I, enhancing transcription On at high frequencyC protein + arabinose bound to I and cAMP + CAP boundto I, enhancing transcription

  22. TrpOperon

  23. trpA Tryptophan Operon

  24. The Regulatory protein of Trp Operon

  25. Control of Trp Operon: ON

  26. Control of Trp Operon: OFF

  27. Attenuation A novel means of controlling gene expression discovered byCharles Yanofsky and his colleagues while studying trpoperon The Observations • Mutants with deletions between the operator and the gene for the first enzyme (trpE) showed increased production of trp mRNA • 5’ end of the trp mRNA revealed the presence of a leader sequence of 162 nucleotides before the initiation codon of trpE. • Mutations that enhanced trp mRNA level mapped in this leader region Nonmutants (wt) produced a transcript consisting of only the first 130 nucleotides of the leader when the tryptophan level was high However, 7000 nucleotide full length trp mRNA including the entire leader sequence was produced when tryptophan was scare

  28. Features of the 5’ UTR • Contains complementary sequences that can form hairpin structures when transcribed into RNA • Codes for a stretch of U nucleotides that can act as a termination signal after a hairpin structure • Codes for several Trp codons as part of an unstable protein product

  29. Termination signal due to hairpin formed by 3+4 pairing followed by string of uracils No terminationsignal formed Alternative RNA Structures from 5’ UTR Formation of termination signal depends on level of tryptophan carried by tRNA in the cell.

  30. AttenuationPremature Termination of Transcription Ribosome translates trp codons, preventing 2+3 pairing 3+4 pairing forms terminator

  31. Antitermination Ribosome stalls at trp codons,allowing 2+3 pairing Transcription continuestoward trp E, D, C. B, A

  32. Summary of Trp Operon Regulation OnTrp repressor inactive Lack of attenuation leads to high rate of mRNA production OffTryptophan + repressor = Active repressor Reduction of mRNA production by attenuation

  33. Thank You

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