Lecture 2: Regulation of Genome Expression

What are the two transcriptional regulation and genetic switches for prokaryotics

• Trp Operon

• Lac Opero

Genomes

• what does it encode

• what are they mainly made out of

  • What is the exception (what are they made out of)

• How are the genome information released out of it? (What is this called?)

• encode info to construct and maintain an organism

• most genomes made out of DNA

  • exception: viruses (not normally considered living things; not made up of cells and need to infect to replicate) - have RNA genomes instead

• genome expression - the release of the biological information stroed in the genome

  • aside: genome itself doesn’t do anything

What is the first product of genome expression? (what is it called?)

• what does it consist of (main def)

What is a DNA microarray - what exactly are you looking it

• how do you read it

• What are the rows

• what are the columns

• what does the red indicate

• What does the green indicate

• What goes the black regions indicate

How often are they used, what else are they using?

• the transcriptome

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

• Image: a DNA microarray (and snapshot of the transcriptome - looking at RNA)

  • Rows = the gene types

  • Column= the different samples

  • Red = lots of DNA from that particular gene

  • Green = very little RNA

  • black: medium amount of DNA from that particular gene

• These days, fewer researchers are using DNA microarrays for transcriptome analysis

  • More are using RNAseq, also done in labs.

How is the transcriptome maintained?

• by the process of transcription

What is the second product of genome expression

• what is it

• what does it define

• How is it maintained

• What snapshot can we use to read this

  • How is it being read

• the proteome

  • the collection of proteins in a cell

  • defines the biochemical functions of the cell

  • Maintained by the process of translation

• 2D gle electrophoresis - gives a snapshot of the proteome

  • Row: the weight of the molecule

  • the column: the isoelectric point (e.g. like polarity)

• blobs not normally coloured in for you - blobs are the proteins

  • no blocks, mean the protein of that type does not exist

  • blue unique proteins

  • larger proteins = more

What is the central dogma of molecular biology

• DNA - genome → RNA - transcriptome → Protein - proteome

Genome expression

• what does all cell types have the same genome

• number of gene in a human

• how much expressed at a time

different cell types contain the same genome

• 25,000 genes

• 30-60% expressed at a time

• expression varies

Regulation of genome expression

• state the main sections and their main definitions

• Genome: DNA

• Transcriptome: the RNA

• Proteome: the proteins

• the bolt = places where we can do something to the DNA

• Interactome: All the protein=protein interactions for the arrows

• Metabolome: All the small molecule metabolites (all the other stuff)

Transcriptional regulation

• crucial for (2 things)

• defining cell types

• responses to extracellular stimuli

Recall

• What enzyme transcribes the RNA

• enzyme: RNA polymerase

• read 3’ to 5’ - the template strand

• make 5 to 3’ - the RNA strand (the sugar phosphate brand)

Recall

• prokaryotic transcription

• what is the promoter

• RNa polymerase zore enzyme

• promoter is region of DNA positions the polymerase and indicates the transcription start site

  • Indicate, is not included in the start site

• Forms the RNA polymerase holoenzyme

Review of transcription

• difference efficiencies

  • Reason: regulated by gene regulatory proteins (transcription factors)

    • Indicates if you make more of the gens or less

• TF worksby binding to regulartory regions of DNA (cis elements)

  • Cis elements = DNA sequences on the same DNA double helix as the gene (where it is located on the genome )

Types of TF

• ON = positive regulators = activator - e.g. make more RNA

• OFF = negative regulators = repressors

E.coli model

features

• unicellular prokaryote

• one choroomsome of circular DNA

• encode 4300 proteins

• geners are transcriptionally regulated by food availability

Prokaryotic feature

• Multiple genes transcribed into a single RNA molecule

  • operon: the DNA region (it’s under the control of one promotor)

The trp (trytophan) operon

• features

• five genes

• encodes enzymes for tryptophan biosynthesis

• transcription regulated by a single promoter

• the operator, provides the start of the RNA synthesis, which then makes the series of enzymes (different enzymes, different) required for tryptophan biosynthesis

Trip operon promoter protein-bound states

• binding by RNA polymerase

  • Trap gene expression is on

• Bound by the tryptophan repressor protein (blocks the promoter access for DNA polymerase for transcription)

  • Gene expression is off

• If the trip repressor binds to a specific DNA sequence of the promoter called the operator

• A cis regulatory sequence

• Numbering for sequencing

  • + is the RNA nucleotides being made

  • - before where the transcription begins

How blocking with repressor works?

• rnA polymerase cannot bind

• Negatively regulates the trap expression

• the repressor DNA-binding activity is still regulated, the repressor must still binds two molecules of tryptophan to bind to DNA

  • Tryptophan is high

    • The tryptophan binds to the repressor, the repressor is activated, binding to the DNA-binding site, preventing the transcription with RNA polymerase

    • Operon is off

  • Tryptophan is low

    • The repressor in inactive

    • The RNA polymerase binds to make e.coli

    • 
      

What is a helix-turn-helix?

• Helix-turn-helix: structural pattern in many proteins that bind to DNA

What does tryptophan repressor have, and why is it important

• tryptophan repressor contains a helix-turn-helix which can bind and recognize DNA sequences

  • BindS in the major grooves of the DNA double helix

  • The binding of tryptophan induces the conformational change and protein can then fit into the major groove

The Lac Operon

• three characteristics of the lac operon

• three genes requires the transport of lactose into the cell and for its catabolism (break down and use for energy)

• Enables use of lactose into the absence of glucose

• Dual regulation: both positive and negative control

Dual regulation: two proteins involved

• Activator: Catabolite activator protein (CAP)

  • Promotes Lac expression: low glucose/high lactose

• repressor: lac repressor protein

  • Inhibited lac expression: low lactose

Three major rules about lac operon (when to use the lac operon)

• E.coli’s first choice is to use glucose

• When there is low glucose and high lactose, it will then use lactose. Both those conditions must be true to use lactose

• The lac operon is what is turned on, when it wants to use lactose

Where does the two proteins binds?

• the cis-regulatory sequence

What happens when lactose is low and glucose is high

• lactose is low, lac expression is bound to the operator

• Lac operon gene expression is off

• Increased lactose will remove the repressor from the operator

Low glucose, high lactose - process with the lac operon

• Is the operon on yet?

• Direct relationship: an increase in lactose increase the levels of allolactose, which requires B - galactosesidase

• The allolactose will bind to the lac operon

  • The lac operon will undergo conformational change And decrease its’s DNA-binding activity, which release the lac operon

• However, the lack operon gene expression is still OFF

What does the lac operon need to activate it’s gene expression

• requires the activator, because the RNA is not good enough to begin get transcription

• Requires the CAP to be bound, that contains the helix]-turn-helix DNA binding domain

How is CAP DNA-binding activated?

• activated by low glucose

• A decrease in glucose levels increase cyclic AMP (cAMP), known as the signalling molecule

  • Decrease of glucose = more cAMP

  • Increase glucose = less cAMP

• cAMP binds to the CAP protein

  • Conformational change

  • Increases the DNA-binding activity

  • The CAP proteins binds to the CAP-binding site

• CAP will recruit the RNA polymerase to the Lac promoter

Quick review

Increase lactose, increase allolactose = allolactose binds to the repressor, the repressor does not bind to DNA, the RNA pol can bind

Decrease glucose, increase in cAMP, cAMP binds to CAP, CAP binds to DNA, RNA pol. Can bind