Bio230 1st half

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

• 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 info 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 information 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)

How to have various cells from a single genome?

A cells have same genome.

Differences in genome expression!

Genome size, how many genes expressed, what type, regulation

• Human genome ~25 000 genes

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

• Expression 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 of gene regulatory proteins (bind to where? pos/neg)

Gene expression regulated by gene regulatory proteins (transcription factors)

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

.

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 (how many promotors?)

• Five genes

• Encode enzymes for tryptophan biosynthesis

• Transcription regulated by a single promoter

The Tryptophan (Trp) Operon Promoter... (repressor binds to what?)

...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

repressor binds to specific DNA sequence of promoter = operator

The Tryptophan (Trp) Operon Promoter binding (does what and needs what?)

Tryptophan repressor binding blocks promoter access

• RNAP cannot bind

• negative regulation

.

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

Helix-turn-helix (binds to what, trp repressor)

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

.

Tryptophan binding to repressor induces

➢ Conformational change

➢ Protein fits into the major groove

Example 2: The Lac Operon (has what and does what?)

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 is what?

1) Activator: Catabolite Activator Protein (CAP)

• Low glucose/High lactose

.

2) Repressor: Lac Repressor Protein

• Low lactose

3 steps for turning ON Lac operon

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

.

glucose lactose high = no

glucose high lactose low = no

glucose low lactose low = no

glucose low lactose high = YES

The Lac Operon Schematic (first operon encodes what)

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

• cis-regulatory sequence for CAP

• lac operator for lac repressor

How is the Lac Operon Regulated? Lactose low

Lactose 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 (allolactose)

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 and positive regulation, bound repressor/activator

Negative regulation: bound repressor prevents transcription

• ligand binds to remove protein

• ligand binds to allow protein binding

.

Positive regulation: bound activator promotes transcription

• ligand vinds to remove protein

• ligand binds to allow protein binding

To summarize negative/positive regulation, competition vs recruiting (ProK) (recruits to where?)

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

.

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

Prokaryotic Gene Regulation: location of gene regulatory elements

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 (kissing) (NtrC)

Some regulatory elements distant from start site

• 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 — 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 (4 types)

Positive feedback loops can be used to create cell memory

1. Parent cell: gene OFF

2. Transient temporary signal: gene ON

3. Positive feedback loop

4. 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: 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 (attenuates before?) (ribo where?)

Riboswitch: 5’ untranslated region (UTR)

• start site of transcription not start site of translation

.

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 — Types of RNA and 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 (TATA box?)

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

Details on eukaryotic transcription

• RNA polymerase II

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

• NO operons

• DNA packaged into chromatin

• Transcriptional activation requires many gene regulatory proteins

Regulatory vs general EUK transcription factors

General transcription factors — needed for any type of transcription

Gene regulatory proteins — activators or repressors

Eukaryotic transcription review — mediators

Intermediate between regulatory proteins and RNA polymerase

EUK gene regulatory proteins (how many, distance)

Eukaryotic gene expression — many regulatory proteins (~2000 encoded by the human genome): activators & repressors

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

• One mechanism is DNA looping

Eul Gene regulatory proteins often function as protein complexes (coactivators/corepressors)

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

Modular design of eukaryotic 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? (3 what?)

Attract, position, and modify:

• General transcription factors

• Mediator

• RNA polymerase II

.

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 (transcription machinery? Activators do what?)

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

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

Nucleosome removal requires cooperation with histone chaperones and chromatin remodelling complex (ATP)

• 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

4) Activator Proteins and Histone Modifying Enzymes — Histone code (what enzymes?)

Histone modifying enzymes produce specific patterns of histone modifications — “histone code”

• phosphorylation (enzyme: kinase)

• acetylation (acetyltransferase)

• methylation (methyltransferase)

Histone modifications occur on specific amino acids of histone tails

The Histone Code: Writer

Specific modifications to histone tails by histone modifying enzymes (writers)

• N-term of histone subunits gets modified

The Histone Code: Reader

Code “reader” proteins can recognize specific modifications and provide meaning to the code

• gene silencing

• gene expression

• chromosome inactivation

Human Interferon Gene Regulation As A Histone Code Example — steps 1 & 2 (started by?)

Step 1: Activator protein binds to chromatin DNA and attracts a histone acetyltransferase (HAT)

Step 2: HAT acetylates lysine 9 of histone H3 and lysine 8 of histone H4

Human Interferon Gene Regulation As A Histone Code Example — steps 3 & 4

Step 3: Activator protein attracts a histone kinase (HK)

Step 4: HK phosphorylates serine 10 of histone H3. Can only occur after acetylation of lysine 9

Human Interferon Gene Regulation As A Histone Code Example — step 5

Step 5: Serine modification signals the acetyltransferase (HAT) to acetylate lysine 14 of histone H3

The histone code for transcription initiation is written

Human Interferon Gene Regulation As A Histone Code Example — step 6 (TFII…and c?)

Step 6: TFIID and a chromatin remodeling complex bind to modified histone tails (and TATA box) and initiate transcription

Eukaryotic Gene Regulation: Repressor Proteins (has what domains?)

Transcriptional Repression:

• Unlike prokaryotes, eukaryotic repressor proteins rarely compete with RNA polymerase for access to DNA

• variety of mechanisms to inhibit transcription

• Repressor has DNA binding domain and repression domain

Eukaryotic Gene Regulation: Repressor Proteins — 1

Competitive DNA binding

• DNA sequence for activator overlaps with sequence for repressor

Eukaryotic Gene Regulation: Repressor Proteins — 2

Masking activation domain of activator

Eukaryotic Gene Regulation: Repressor Proteins — 3

Direct interaction with general transcription factors (TFII)

• stops general transcription factors

Eukaryotic Gene Regulation: Repressor Proteins — 4

Recruitment of chromatin remodelling complexes

• rewrapping DNA

Eukaryotic Gene Regulation: Repressor Proteins — 5

Recruitment of histone deacetylases

• writing on tails of histones

Eukaryotic Gene Regulation: Repressor Proteins — 6

Recruitment of histone methyltransferase

• writing on tails of histone

A More Complex Example of Transcriptional Repression (guided by what)

Guided by gene regulatory proteins, histone “reader” and “writer” proteins can establish a repressive form of chromatin

• writer recruits reader

• reader recruits writer

• repeat

• can also write directly on DNA

• Histone code can spread!!!

A More Complex Example of Transcriptional Repression — barrier sequences (DNA more?)

Spreading the histone code along chromatin carried out by reader-writer complexes

• barrier sequences (border sequencess) to stop reader-writer

• DNA more compact/condensed, transcription factors and RNAP cant access

A More Complex Example of Transcriptional Repression — DNA methylase (methylates what)

DNA methylase enzyme is attracted by Reader and methylates nearby cytosines in DNA (AGCT and methylated C)

• DNA methyl-binding proteins bind methyl groups and stabilize structure

• methylation and therefore gene expression patterns can be inherited (epigenetic inheritance)

Various Techniques Allow for Transcriptome Analyses

• Analysis of a large set of cellular transcripts

• Provides a “signature” of cell type

  • DNA microarray

Transcriptome Analyses is Powerful! (for seeing what states?)

Provides a “signature” of cell state

➢ response to extracellular stimuli

➢ disease states e.g., cancer

Transcriptome Analyses provides a “signature” of cell state

Compare transcriptomes of:

• Model cell for cancer

• Cancer cells from patient

• Normal cells from patient

• Chemically treated — potential cancer drugs

Transcriptome Analyses is Powerful! Previous example drugs

IC50: Concentration of compound required to inhibit cell proliferation by 50%

• lower better, stronger

A Review of RNA Processing (euk coupling vs prok coupling?)

Eukaryotic RNA processing is tightly coupled to transcription:

1. covalent modifications of RNA ends (5’ cap, poly A tail)

2. removal of intron sequences

(prokaryotes translation is coupled to translation, start translation before transcription ends)

A Review of RNA Processing: RNA Capping

Addition of a modified guanine nucleotide to the 5’ end of pre-mRNA (3 enzymes involved)

Cap bound by cap-binding complex (CBC)

What does 5’ cap do after exporting?

1. helps RNA processing and export from nucleus

2. important role in translation of mRNA in cytosol

3. protects mRNA from degradation

   • 5’ exonuclease (5’ to 3’ exonuclease)

   • cuts one nucleotide at a tide

   • cuts 5’ to 3’ phosphodiester bonds

   • so weird that exonucleases cannot cut anymore

A Review of RNA Processing: RNA Splicing — introns and exons

Coding sequences (exons) and non-coding sequences (introns)

• both are transcribed into RNA, but introns are removed by RNA splicing

RNA Splicing — alternative splicing does what? (percentageeeeeee)

~75% of human genes produce multiple proteins — increases coding potential of genomes

RNA Splicing — Spliceosomes and exon junction complexes (EJCs)

Spliceosomes — Enzyme complex made up of RNA and proteins

Sites of proper splicing bound by exon junction complexes (EJCs)

• serve as a marker for properly spliced RNA

Alternative Splicing Can Be Regulated — negative control

Default: there is splicing

Repressor on: no splicing

Alternative Splicing Can Be Regulated — positive control

Default: no splicing

Activator on: splicing