Bacterial and eukaryotic gene expression regulation

Why do cells regulate transcription in bacteria?

-Bacteria respond to changes in environmental conditions (such as presence of nutrients or change in temperature)

• Most (but not all) gene regulation in bacteria occurs at the level of transcription initiation

• Only about 5% of the genes are actively transcribed at any one time (in E. coli) bacteria

What are the principles of gene expression regulation?

• DNA-protein interactions- Transcription factors (repressors and activators) are DNA-binding proteins

• Protein-protein interactions- Transcription factors interact with each other and/or RNA polymerase

• RNA interactions- Small regulatory RNAs or (untranslated)

regions of mRNAs regulate expression

• Response to the environment- Nutrients, etc., in unicellular organisms Hormones, etc., in multicellular organisms

How do proteins bind DNA?

DNA Binding proteins have high specificty( multiple weak interactions) and bind to the alpha helix in the major groove in dna

What are the functions of transcription factors?

• Repressors: prevent RNA polymerase from binding promoter or initiating transcription

- if tryptophan high than it represses

• Activators: facilitate RNA polymerase binding or initiation

★Bacterial transcription factors often interact with small molecules or are post-translationally modified in

response to environmental conditions

What is an operator

Dna sequence downstream from promoter where transcriptional factors, activators and repressors bind to it.

What are the differences

and similarities between transcriptional activators and repressors?

-Both bind to operator

-Both regulate gene expression

-repressor prevents RNA polymerase from binding

promoter or initiating transcription

-activators Facilitate RNA polymerase binding or

initiation

What is an operon?

• Two or more genes transcribed together from the same promoter

• Produce a polycistronic mRNA

• Allow coordinate regulation of functionally related genes

What is the mechanism of transcriptional regulation by repressors?

-prevent rna polymerase from binding to promoter or initiating transcription

- in high amounts of tryptophan the tryp repressor will bind to operator and prevent rna synthesis

What is the mechanism of transcriptional regulation by

activators?

-they facilitate rna polymerase binding or intiation

What is the lac operon and why is it regulated?

• Three structural genes involved in metabolism of lactose (milk sugar)

- lacl, lacz, operator

• The Operon turned on when lactose is present and glucose (E. coli's favorite carbon source) is absent

• lacZ: structural gene (in lac operon) encoding β-galactosidase

-Breaks down lactose

• Repressor (transcription factor encoded by lacI) that binds operator

-Inhibits transcription of lac operon

• Operator (adjacent to lac promoter)

-DNA sequence where repressor (LacI) binds

How is the lac operon negatively regulated?

• When Lactose is absent

• Repressor (LacI) binds to operator

• RNA polymerase either cannot bind to promoter or cannot initiate transcription

What is induction?

• Turning the operon on (increasing

transcription)

• Requires the presence of lactose (and

the absence of glucose)

• Inducers:

Physiological: allolactose (formed from

lactose)

Experimental: IPTG (lactose analog that

cannot be metabolized)

What is the mechanism of induction?

• Inducer binds to repressor (LacI)

• Repressor changes shape (allosteric

interaction)

• Repressor/inducer complex cannot bind

operator

• lac operon transcribed

- Lactose present (and glucose absent)

- Repressor does not bind operator

How and why is the lac operon positively regulated?

Glucose is the favorite sugar (carbon

source)

• There is no need to use lactose if

glucose is present

• The lac operon (and operons encoding

the catabolism of other sugars) are

kept off if glucose is present

• Catabolite activator protein

• Receptor for cAMP in bacteria

• Also called CRP (cAMP receptor protein)

• cAMP-CAP complex binds near promoter

and facilitates RNA polymerase binding

• Lactose present

• Glucose absent (cAMP level high)

What is the role of cAMP?

Signals low glucose

nucleotide

Binds to CAP

Which RNA interactions

affect gene expression and how?

RNA to RNA interactions- will not encode protein unless there is an increase in temperature exposing ribosome binding site

RNA to protein interactions- will encode protein unless their is a translational repressor protein binds to to start codon aug

What are the differences and similarities between gene expression in bacteria and eukaryotes?

Prokaryotic gene expression is primarily controlled at the level of transcription.

Eukaryotic gene expression is controlled at the levels of epigenetics, transcription, post-transcription, translation, and post-translation.

Prokaryotic gene expression (both transcription and translation) occurs within the cytoplasm of a cell due to the lack of a defined nucleus; thus, the DNA is freely located within the cytoplasm.

Eukaryotic gene expression occurs in both the nucleus (transcription) and cytoplasm (translation).

What are the roles of the three eukaryotic nuclear RNA polymerases?

Three RNA polymerases catalyze transcription

• RNA polymerase I:

Pre-rRNA

• RNA polymerase II:

Pre-mRNA (protein-coding),

miRNA, some small RNAs

• RNA polymerase III:

-involved in transcription of small rnas

- is the primary enzyme complex involved in prokaryotic DNA replication.

-high processivty, a dimer bc beta subunit tethers dna p3 to template

Pre-tRNA, pre-5S rRNA, small structural RNAs

What is the difference between a cis-acting factor and a trans-acting factor?

• Cis-acting: affects genes only on the same DNA (or RNA) molecule

➡ DNA (or RNA) sequence

• Trans-acting: affects genes on same or different DNA molecules (diffusible)

➡ Protein (or small regulatory RNA)

What is a cis-acting regulatory element?

Transcription factor binding sites

• Promoter-proximal elements

-Within 200 base pairs of start site

• Enhancers (and silencers)

- 200 to >10,000 base pairs from start site

-Upstream or downstream

What are transcription factors and what two domains do they have?

• Bind to promoter-proximal elements and

enhancers (or silencers)

• Activators stimulate transcription

• Repressors inhibit transcription

• Composed of two parts (modular):

- DNA-binding domain

- Activation (or repression) domain

Most genes are regulated by multiple

transcription factors

Why is transcriptional

regulation important for cell differentiation?

• Properties of a cell are determined by which genes are expressed

• Which genes are expressed are determined by which transcription factors are active