Gene Regulation - Lecture notes 16 PDF

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Dr. Beal - Lecture Notes 16 Fall 2019 - Gene Regulation...

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Gene Regulation ● ● ● ● I. ● ● A. ●

Gene Expression is regulated All cells have genes but not all genes are expressed all the time Constitutive genes are always expressed Some important genes are not always expressed

Prokaryotic Gene Regulation (SEQ, HD lac operon; CC positive and negative regulation) Mainly at transcription DNA->RNA Lac operon In e-coli (modeled) ○ Normal gut bacteria- human intestine ○ Can adjust based on host diet (can change enzymes to digest food) ○ Humans drink milk -> E. coli digests lactose ● Induction: turning on gene expression ● Inducer: compound that stimulates gene expression ● Inducible enzyme: coded for by inducible gene ○ Produced in response to inducer ○ Contrast with constitutive enzymes ● Operon ○ Complex DNA consisting of: a group of genes with related functions; regulatory DNA sequences ○ Only found in prokaryotes ● Ex: lac operon contains ○ Promoter: RNA polymerase binding site, not transcribed (states to start transcription right next to it) ○ Operator: “switch” sequence for gene expression ○ 3 genes: lacZ, lacY, lacA ○ In order: promoter, operator, 3 genes (Z,Y,A) B. Negative regulation of lac operon ● No lactose -> operon off, no expression ● With lactose -> operon expressed ● Involves repressor ○ Made by repressor gene: codes for repressor protein- down-> regulates gene expression ○ Always on: constitutive expression ○ Not part of operon (located before operon -> repressor promoter and gene) ● Lac repressor activity ○ No lactose: repressor binds to lac operator ○ RNA polymerase (RNApol) can bind to promoter, but cannot transcribe genes ○ NO lac operon expression ● Lactose affects lac expression ○ Lactose enters cell, converted to allolactose (lactose isomer) ○ Binds to 2nd site on repressor protein ○ Inactivated by changing structure -> cannot bind operator

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Allosteric regulation ■ Allolactose is a non-competitive inhibitor of repressor protein ● Allolactose is the inducer ○ Induces lac operon genes by inactivating repressor protein ○ Allows transcription to occur ○ Result: lows levels of lac expression ○ What is transcribed: ■ Long mRNA with all 3 genes ■ Each gene has own start and stop codons ■ Translated as 3 polypeptides ■ Takes place in nucleoid ■ Transcription and translation happen at the same time in prokaryotes so nucleus and mRNA is synthesized and translated simultaneously C. Positive control of the Lac operon ● Positive control: regulation by an activator that binds DNA to stimulate transcription- HIGH expression when active ○ Independent of Lactose ○ Lactose can be used for E but glucose is preferred ○ If glucose is present, use that first & Lac operon expression responds to lactose and glucose levels ● Lac promoter is inefficient ○ Only low levels of expression bc promoter has low affinity for RNApol ○ Solution: CAP (catabolite activator protein) ■ Increases affinity of promoter for RNApol ■ CAP is inactive alone -> activate when bound to cAMP (cyclic AMP-adenosine monophosphate) ● Effects of CAP-cAMP: low glucose causes cAMP to increase ○ CAP-cAMP binds CAP-binding site next to RNApol binding site ■ Bends double helix & increases RNApol binding ■ Positive control -> increases expression ○ Positive lac regulation: CAP binds to cAMP and has an affinity for RNApol so result is high levels of lac expression ● Lac Operon summary ○ Neg control: repressed in the absence of lactose ○ Pos control: induced in the absence of glucose ● Lac operon activity: 4 situations ○ High glucose/no lactose- no CAP, repressed, no expression ○ No glucose/no lactose- CAP, repressed, no expression ○ High glucose/high lactose- no CAP, induced, low expression ○ No glucose/high lactose- CAP, induced, high expression

II. Eukaryotic Gene Regulation (SEQ, HD, CC eukaryotic gene regulation) A. Introduction

● Eukaryotic cells also respond to environment ● Multicellular: allows for specialization and organization ● Achieved through differential gene expression ● Unlike in prokaryotes, every level is regulated B. Regulation of chromatin structure ● Euchromatin: loosely packed; genes can be active ● Heterochromatin: densely packed; no gene expression ● Can be regulated by DNA methylation ● Also via histone modification: ○ methylation (hydrophobic) inhibits transcription ○ acetylation (hydrophilic) promotes transcription C. Regulation of Transcription initiation ● Generally more complex than prokaryotes ● Involves DNA sequences and transcription factors-proteins that bind DNA ● Promote or inhibit transcription initiation ● Summary: ○ Dna packaging: histone and/or DNA modification ○ DNA control elements D. Post-transcriptional regulation (manipulate RNA) ● Length of poly-A tail affects the level of expression ● Alternative splicing ○ Different polypeptides from the same gene E. Post-translational regulation ● Polypeptides often processed -> final protein ● Must be folded correctly ● Often chemical modifications Summary: ● More complex than prokaryotes ● Can involve: DNA packaging, transcription regulation, post-transcriptional regulation, posttranslational regulation...