Bio230 1st half

Lecture 1: Regulation of Genome Expression

How can the genome create and maintain a living organism?

The tree of life has three primary branches...

Bacteria, Archaea, Eukaryotes

• archaea closer related to eukaryotes, but still prokaryote

Prokaryote and Eukaryote

1) Prokaryotic organisms

• eubacteria and archaea

• single-celled

• no nucleus and organelles

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2) Eukaryotic organisms

• plants, fungi, animals

• single or multi celled

• nuclei and organelles

Prokaryotic cell

• nucleoid

• SMALLLER

Eukaryotic cell

• LARGER

Prokaryotic and Eukaryotic Cells Live Together in the Human Body

Microbiota: microorganisms of a site (bacteria, archaea, fungi, protists, and viruses)

• reside on skin, lungs, gut, etc

• ratio of microbial:human cell is at least 1:1

• important role in health

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Microbiome: combined genomes of microbiota

• 200X more microbial genes than human in human body

Lecture 2: Genomes

• All known life forms possess a genome

• Encodes the information to construct and maintain organism

• Most genomes are made of DNA

  • except some viruses have RNA genomes (not living, no cells, some have DNA)

• Release of the biological information stored in the genome requires genome expression

Genome Expression: The Transcriptome

First product of genome expression is transcriptome

• repertoire of RNA molecules in a cell at a particular time

• maintained by the process of transcription

DNA microarray: snapshot of transcriptome

How to read

• rows = genes

• columns = samples

• red = lots of RNA

• black = in between

• green = minimal RNA

RNA sequencing more popular now

Genome Expression: The Proteome

The second product of genome expression is the proteome

• Collection of proteins in a cell

• Defines the biochemical functions of the cell

• Maintained by the process of translation

2D gel electrophoresis: snapshot of the proteome

How to read

• y-axis = molecular weight

• x-axis = isoelectric point (pH)

• splotches = proteins

• bigger blob = more protein

not normally colour coded

• red = common proteins (both has)

• blue = unique proteins

• diff cells can have same protein at different amounts

• or diff proteins

‘Central dogma’ of molecular biology

Genome (DNA) ➔Transcriptome (RNA) ➔Proteome (Protein)

Genome Expression — how to have various cells?

Different cell types of a multicellular organism contain the same genome

How do we produce different cell types?

Differences in genome expression!

Genome Expression — how to have various cells? — cont

Human genome ~25 000 genes

At any one time only 30-60% of genes expressed

Expression of almost all genes varies from one cell type to another

Genome expression is regulated at many steps from DNA to protein

Regulation of Genome Expression — red lightning = ways of regulating

Interactome

All protein-protein interactions

Metabolome

All small molecule metabolites

• nutrients

• waste

• etc

Regulation of gene expression is crucial for:

Defining Cell Types (multicellular organisms)

Responses to extracellular stimuli (both multicellular and unicellular organisms)

A Review of Prokaryotic Transcription...

Sigma factor — binds to promotor

• small space in RNA core enzyme before active site for sigma factor

Holoenzyme = sigma factor + RNA core enzyme

Transcriptional Regulation: Overview

Gene expression in prokaryotes & eukaryotes is regulated by gene regulatory proteins (transcription factors)

• Bind specifically to regulatory regions of DNA (cis elements — on the same DNA double helix as gene, trans is different double helix)

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Gene regulatory proteins can turn genes:

ON = Positive regulators = activators

OFF = Negative regulators = repressors

E. coli:

• unicellular prokaryote

• one chromosome of circular DNA encodes about 4300 proteins

• many genes are transcriptionally regulated by food availability

Prokaryotic operons

Multiple genes can be transcribed into a single RNA molecule

• under control of one promotor

Example 1: The Tryptophan (Trp) Operon

• Five genes

• Encode enzymes for tryptophan biosynthesis

• Transcription regulated by a single promoter

The Tryptophan (Trp) Operon Promoter...

...has two potential protein-bound states:

1. Bound by RNA polymerase — Trp gene expression ON

2. Bound by tryptophan repressor protein — Trp gene expression OFF

tryptophan repressor binds to specific DNA sequence of promoter = operator

The Tryptophan (Trp) Operon Promoter binding

Tryptophan repressor binding blocks promoter access

• RNAP cannot bind

• negative regulation

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Repressor DNA-binding is regulated

• must bind TWO molecules of tryptophan

• repressor and operator — control tryptophan biosynthesis according to availability of free tryptophan

The Tryptophan Operon: Summary

The Structure of the Tryptophan Repressor...Helix-turn-helix

Tryptophan repressor contains a helix-turn-helix DNA binding motif (most common DNA-binding motif)

• structural category in many DNA-binding proteins

• bind to major groove in double helix

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Tryptophan binding to repressor induces

➢ Conformational change

➢ Protein fits into the major groove

Example 2: The Lac Operon

E. coli Lac operon:

• 3 genes required for transport of lactose into cell for catabolism (breaking down for energy)

• enables use of lactose in absence of glucose

• positive and negative control

Lac Operon activator/repressor

1) Activator: Catabolite Activator Protein (CAP)

• Promotes Lac expression: Low glucose/High lactose

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2) Repressor: Lac Repressor Protein

• Inhibits Lac expression: Low lactose

3 rules for Lac operon ON

1. E.coli first choise is glucose

2. Low glucose, high lactose = use lactose (BOTH conditions must be true)

3. lac operon turned on to use lactose

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glucose lactose high = no

glucose high lactose low = no

glucose low lactose low = no

glucose low lactose high = YES

The Lac Operon Schematic

1st gene of Lac operon encodes β-galactosidase; breaks down lactose to glucose and galactose

How is the Lac Operon Regulated? Lactose low

When lactose levels are low, the lac repressor is bound to the operator

• Lac operon gene expression is OFF

• Increased lactose removes the repressor from the operator...

How is the Lac Operon Regulated? Lactose high

Increases in lactose increase levels of allolactose, related to lactose; requires β-galactosidase

• allolactose binds to lac repressor

  • conformational change, decrease DNA-binding activity, release from operator

How is the Lac Operon Regulated? activator

Why is an activator needed?

• RNA polymerase binding is inefficient to the Lac promoter

• Efficient RNA polymerase binding to Lac promoter requires CAP to be bound

• CAP contains a helix-turn-helix DNA binding domain

How is the Lac Operon Regulated? Glucose low

Low glucose —→ high signaling molecule cyclic AMP (cAMP)

• cAMP bind to CAP protein

  • conformation change, increases DNA-binding activity

  • binds to CAP binding site

CAP recruits RNA polymerase to the Lac promoter

Prokaryotic Gene Regulation

Negative regulation: bound repressor prevents transcription

• ligand binds to remove protein

• ligand binds to allow protein binding

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Positive regulation: bound activator promotes transcription

• ligand vinds to remove protein

• ligand binds to allow protein binding

To summarize:

• Negative regulation: Competition between RNA polymerase and repressor protein for promoter binding

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• Positive regulation: Activator protein recruits RNA polymerase to the promoter to activate transcription

Prokaryotic Gene Regulation: start sites

Gene regulatory elements are typically close to the transcriptional start site of prokaryotic genes

But CAN be:

• far upstream

• downstream

• within gene (introns, eukaryotes only)

Some regulatory elements are distant

Some regulatory elements are distant from the transcriptional start site and influence transcription

• DNA looping to allow interaction between protein and RNAP

• “kissing”

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NtrC protein is a transcriptional activator

DNA looping allows NtrC to directly interact with RNA polymerase — activate transcription from distance

A Genetic Switch Example from Bacteriophage Lambda

Virus that infects bacterial cells

• Positive and negative regulatory mechanisms work together to regulate the lifestyles of bacteriophage lambda

Bacteriophage lambda can exist as one of two states in bacteria

Prophage pathway: integration of DNA into host chromosome, dont do much (favourable conditions)

Lytic pathway: synthesis of many copies and lyses (host cell is damaged)

• Two gene regulatory proteins responsible for initiating this switch — repress each other’s synthesis

Two gene regulatory proteins are responsible for initiating the switch between prophage and lytic pathways

Lambda repressor protein (cI) and Cro protein

• repress each other’s synthesis, giving rise to the two states

State 1: prophage state

Lambda repressor protein made

• occupies operator

• blocks synthesis of Cro

• activates own synthesis (both repressor and activator)

• most viral DNA not transcribed

State 2: Lytic state

Lambda Cro protein is made

• occupies operator

• blocks synthesis of lambda repressor

• allows own synthesis (NOT an activator)

• most Viral DNA is extensively transcribed

• DNA replicated, packaged, new bacteriophage released by lysis

What triggers the switch between prophage and lytic states?

Host response to DNA damage!

• induction event — switch to lytic state inactivates repressor

• bac activates DNA repair proteins

• virus detects, something wrong, LYSE TIME

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Under good growth conditions, lambda repressor protein turns off Cro and activates itself in a positive feedback loop (prophage state)

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The prophage-lytic control is an example of a transcriptional circuit

Transcriptional Circuits

Positive feedback loops can be used to create cell memory

Parent cell: gene OFF

Transient temporary signal: gene ON

Positive feedback loop

Progeny continues to have feedback loop in abscence of initial signal

• how specialized cells keep its memory

Feed-forward loops can measure the duration of a signal

BOTH A and B required for transcription of Z

• needs duration of A and B to make Z

• need enough of A to make B and to bind to Z operator

Complex transcriptional circuits

Combinations of regulatory circuits in eukaryotic cells to create exceedingly complex regulatory networks

• can construct artificial circuits and examine behavior in cells

• “synthetic biology”

An Example of Synthetic Biology: The Repressilator

The Repressilator: scientists created a simple gene oscillator using a delayed negative feedback circuit

• A: Lac repressor

• B: Tet repressor (response to antibiotic)

• C: Lambda repressor

Predicted: delayed negative feedback give rise to oscillations in gene expression

The Repressilator — how does it work?

1. A expressed

2. B repressed

3. C expressed

4. C represses A expression

5. A repressed

6. B expressed

7. C repressed

8. REPEAT

The Repressilator — real results

• increasing amplitude due to bacterial growth

Transcriptional Attenuation

Premature termination (both prok and euk)

• RNA adopts a structure that interferes with RNA polymerase

• Regulatory proteins can bind to RNA and interfere with attenuation

Riboswitches

Prokaryotes, plants and some fungi also use riboswitches to regulate gene expression

Riboswitches: short RNA sequences that change conformation when bound by small molecule (some do transcription attenuation)

• eg. prokaryotic riboswitch that regulates purine biosynthesis

Prokaryotic riboswitch that regulates purine biosynthesis

Riboswitch: 5’ untranslated region (UTR)

• start site of transcription not start site of translation

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Low guanine levels: Transcription of purine biosynthetic genes is ON

High guanine levels:

• guanine binds to riboswitch

• riboswitch conformational change

• forms transcription terminator

• causes RNAP to terminate (transcription attenuation) before coding sequence

• Transcription of purine biosynthetic genes is OFF

Eukaryotic transcription review — RNA polymerases

Cells produce several types of RNA

• mRNA, rRNA, tRNA (both euk and prok, euk has more)

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Different RNA transcribed by diff RNAP in EUKARYOTES

• Pol I = rRNA

• Pol II = mRNA

• Pol III = tRNA

Prok only have 1 type

Eukaryotic transcription review — General transcription factors

Help position RNAP at eukaryotic promoters

• TF = general transcription factor

• II = RNA pol II

• subunit = D, B, A, etc

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Many euk promoters have TATA box

Eukaryotic transcription review — General transcription factors cont.

• RNA polymerase II transcribes protein coding genes

• Requires five general transcription factors: TFIID, TFIIB, TFIIF, TFIIE, and TFIIH (prokaryotes only need one; σ factor)

• Eukaryotic genomes lack operons

• Eukaryotic DNA is packaged into chromatin which provides an additional mode of regulation

• Eukaryotic transcriptional activation requires many gene regulatory proteins

EUK transcription factors regulatory vs general

General transcription factors — needed for any type of transcription

Gene regulatory proteins — activators or repressors

Eukaryotic transcription review — mediators

The mediator acts as an intermediate between regulatory proteins and RNA polymerase

EUK gene regulatory proteins

Eukaryotic gene expression is controlled by many regulatory proteins (~2000 encoded by the human genome): both activators and repressors

Gene regulatory proteins can act over very large distances, sometimes >10 000 base pairs away

One mechanism is DNA looping

Eukaryotic Gene Regulation: Protein Complexes

Eukaryotic gene regulatory proteins often function as protein complexes on DNA

• coactivators and corepressors: assemble on DNA-bound gene regulatory proteins, do NOT directly bind DNA

  • helps with activation/repression

• some proteins can have multiple functions depending on neighboring proteins

Eukaryotic Gene Regulation: Activator Proteins

Modular design:

1. DNA binding domain (DBD) — recognizes specific sequence

2. Activation domain (AD) — accelerates frequency/rate of transcription

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• As long as DBD is bound to DNA, AD can do its job

• Can mix-and-match DBD and AD (evolution, biotech, synthetic biology)

How do activator proteins activate transcription? Outline

Attract, position, and modify:

• General transcription factors

• Mediator

• RNA polymerase II

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Either:

1) Directly by acting on these components

2) Indirectly modifying chromatin structure

EUK Activator Proteins — binding directly

1) Activator proteins can bind directly to transcriptional machinery or the mediator and attract them to promoters (like prokaryotic activators)

EUK Activator Proteins — altering chromatin structure

2) Activator proteins can alter chromatin structure

Nucleosomes and histones

Nucleosomes are the basic structure of eukaryotic chromatin

• ~200 DNA base pairs wound around a histone octamer

  • (H2A, H2B, H3, and H4) x 2

  • one nucleosomes includes linker DNA = 10-80 base pairs

  • DNA wraps around histone 1.7 times = approx 147 base pairs

  • separate from core histone, H1 protein

Nucleosomes pack as compact chromatin fibers

Zigzag model vs Solenoid model

• transcription machinery cannot assemble on promoters tightly packed in chromatin

• activator proteins can alter chromatin structure — increase promoter accessibility

4 major ways activator proteins can alter chromatin PICTURE

1) Activator Proteins and Nucleosome Sliding

• chromatin remodelling complex

Nucleosome structure altered by chromatin remodeling complexes

• ATP-dependent

• increase promoter accessibility

• allows access of transcription machinery to DNA

• “rolls around to make space”

2) Activator Proteins and Histone Chaperones — removing nucleosomes

• Histone chaperones and chromatin remodelling complex (ATP)

Nucleosome removal requires cooperation with histone chaperones

• remove histone core

• transcription machinery assembles on nucleosome free DNA

• OPTIONAL: variant proteins combine to general transcription factors to help with transcription

3) Activator Proteins and Histone Chaperones — histone exchange

• Histone chaperones and chromatin remodelling complex (ATP)

Change histone octamer

• histone variants allow greater access to nucleosomal DNA

• looser binding