Nilansu Das Dept. of Microbiology Surendranath College Operons Nilansu Das Dept. of Microbiology Surendranath College

Group A: Cellular and Molecular Biology 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.

Prokaryotic Gene Regulation Coordinate regulation of genes involved in similar functions

Types of Control Negative Control Product of regulatory gene inhibits transcription Positive Control Product of regulatory gene enhances transcription

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

Types of Operons Inducible Initial condition: OFF Inducer switches operon ON Repressible Initial condition: ON Repressor switches operon OFF

Regulation of the Lac Operon Pcrp crp Pi O I P Lac Z Lac Y Lac A DNA Function Protein Function Pcrp Promoter for crp gene crp Gene for CAP protein Positive regulator Pi Promoter for I gene I Gene for Lac Repressor Negative regulator P Promoter for Structural Genes O Operator Lac Z Gene for B-galactosidase Cleaves lactose Lac Y Gene for Permease Lactose transport Lac A Gene for Acetylase Unknown Structural Genes

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

Regulation of the Lac Operon: Low lactose, High glucose Lac Z Lac Y Lac A P O Pi I Pcrp crp Pol 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. Active repressor crp mRNA I mRNA Inactive CAP protein No mRNA produced No Z, Y, A proteins produced

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

Regulation of the Lac Operon: High lactose, Low glucose Lac Z Lac Y Lac A P O Pi I Pcrp crp Active repressor crp mRNA I mRNA Inactive CAP protein Lactose + Pol Z, Y, A mRNA Transcription Translation B-galactosidase Permease Acetylase + 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. cAMP Active CAP protein

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

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

Summary of Lac Operon Regulation Level of Lactose Level of Glucose Lac Operon Low High Off Off On at low frequency On at high frequency

Mutations of the Lac Operon Pcrp Pi O CRP I P Lac Z Lac Y Lac A Functional genes: I+ P+ O+ Z+ Y+ A+ I+ Functional Repressor Trans-acting I- Non-functional Repressor Is Superrepressor (cannot bind lactose) Is> I+> I- 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).

Mutations of the Lac Operon Pcrp Pi O CRP I P Lac Z Lac Y Lac A Functional genes: I+ P+ O+ Z+ Y+ A+ P+ Functional Promoter Cis-acting P- Non-functional Promoter O+ Functional Operator Oc Non-functional Operator (Operator Constitutive) O- (Operator region deleted)

Mutations of the Lac Operon Pcrp Pi O CRP I P Lac Z Lac Y Lac A Functional genes: I+ P+ O+ Z+ Y+ A+ Z+ Functional B-galactosidase Z- Non-functional gene for B-galactosidase Y+ Functional Permease Y- Non-functional gene for Permease A+ Functional Acetylase A- Non-functional gene for Acetylase A mutation in one structural gene does not affect the production of proteins from the other structural genes.

Lac Operon Mutations I+P+O+Z+Y+ I-P+O+Z+Y+ -- + -- + I+P-O+Z+Y+ B-Galactosidase Permease No lactose Lactose No lactose Lactose I+P+O+Z+Y+ I-P+O+Z+Y+ I+P-O+Z+Y+ I+P+OcZ+Y+ I+P+O+Z-Y+/ I-P+O+Z+Y+ I+P+OcZ+Y-/ I+P+O+Z-Y+ I+P+OcZ-Y+/ I-P+O+Z+Y- IsP+O+Z+Y-/ I-P+O+Z-Y+ I+P-OcZ+Y+/ I+P+O+Z-Y- -- + -- + + + + + -- -- -- -- + + + + -- + -- + + + -- + -- + + + -- -- -- -- -- -- -- --

Arabinose Operon Ara C I O Ara B Ara A Ara D DNA Function Protein Function Ara C Codes for C protein Positive and Negative regulator I Initiator (promoter region) Binds C protein O Operator Ara B Structural gene Ara A Ara D

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

Summary of Ara Operon Regulation Level of Arabinose Level of Glucose Ara Operon Low High Off C protein bound to O and I, Inhibiting transcription Off C protein bound to O and I On at low frequency C protein + arabinose bound to I, enhancing transcription On at high frequency C protein + arabinose bound to I and cAMP + CAP bound to I, enhancing transcription

Trp Operon

Tryptophan Operon Trp R P O Trp E, D, C, B, A 5’ UTR (Leader) trpA DNA Function RNA/Protein Function Trp R Gene for repressor Binds to operator to inhibit transcription P Promoter O Operator Trp E, D, C, B, A Structural genes Enzymes acting in pathway to produce tryptophan. Gene order correlates with order of reactions in pathway. 5’ UTR (Leader) Premature termination of transcription when trp levels are high ‘

The Regulatory protein of Trp Operon

Control of Trp Operon: ON

Control of Trp Operon: OFF

Attenuation The Observations A novel means of controlling gene expression discovered by Charles Yanofsky and his colleagues while studying trp operon 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

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

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

Attenuation Premature Termination of Transcription Ribosome translates trp codons, preventing 2+3 pairing 3+4 pairing forms terminator

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

Summary of Trp Operon Regulation Level of Tryptophan Trp Operon Low High On Trp repressor inactive Lack of attenuation leads to high rate of mRNA production Off Tryptophan + repressor = Active repressor Reduction of mRNA production by attenuation

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