Biol 190 Lec 20

Why do all organisms regulate gene expression?

1. Conserve energy (to only make the needed proteins)
2. Respond to the environment

What is an additional reason why multicellular eukaryotes would want to regulate gene expression?

To regulate development (i.e. cell specialization to turn on the right genes to develop zygotes into specialized cells with defined tasks like the central nervous system)

Why do bacteria have fewer properties they are able to control than multicellular eukaryotes for gene expression?

Bacteria is smaller, typically one cell

What are the two categories of gene expression?

Inducible and constitutive

Describe the first category of gene expression.

Inducible gene expression: expressed only when needed by the cell, involves repressors and activators

Describe the second category of gene expression.

Constitutive gene expression: actively expressed at all times

How is gene expression controlled in prokaryotes/bacteria?

Operon model

What is a regulatory region?

An "on-off switch" that controls a cluster of functionally related genes; part of the operon

Define operator.

Regulatory "on-off switch"; a segment of DNA usually positioned within the promoter; controlled by part of the promoter region

Define operon.

Stretch of DNA that includes the operator, the promoter, and the genes that are under control

Define structural genes.

Genes that build the protein

Do bacterial mRNAs have introns?

No

What does it mean for bacterial mRNAs to be polycistronic?

They encode for more than one protein (e.g. 5 structural genes can encode for 5 enzymes of the same metabolic pathway)

Multiple structural genes are controlled by \____ promoter.

One

Operons can \_____ or \_____.

Build something; Break something down

What are the characteristics of the trp (tryptophan) operon?

1. Synthetic (building) operon- makes tryptophan
2. Active in the absence of tryptophan (amino acid)
3. Repressible operon (naturally on)

What lies upstream of the operon and is not a part of it?

Regulatory gene (e.g. trpR)- undergoes gene expression to make a repressor protein (interacts with the operator)

When does the repressor protein of trpR interact with the operator?

Too much tryptophan (corepressor) present-\> corepressor binds to the trp repressor protein, turning the operon off

What happens when the trp operon is active AKA repressor protein is inactive (not binded to the operator)?

RNA polymerase is able to bind to the promoter, read the structural genes, and make multiple enzymes (polycistronic)

Where is the repressor protein if it is not binded to the operator?

Cytoplasm

What is the importance of the trp operon?

E. coli can synthesize the amino acid tryptophan, but the operon will limit production to only what is needed

Review: When tryptophan is absent, is the repressor inactive/active and is the operon on/off? What about when tryptophan is present?

Trp absent: repressor inactive, operon on; Trp present: repressor active, operon off

What are the characteristics of the lac (lactose) operon?

1. Catabolic operon- breaks down lactose
2. Inactive in the absence of lactose
3. Inducible operon (naturally off)

What is the name of lac operon's regulatory gene?

lacI (Note: I stands for inducible)

If glucose and lactose are both present, which one will bacteria eat first (prefer)?

Glucose

What happens when the lac operon is inactive AKA repressor protein is active (binded to the operator)?

RNA polymerase is unable to bind to promoter; no transcription-\> no protein

When does the repressor protein of lacI stop interacting with the operator?

Low glucose and allolactose (inducer) present; inducer inactivates the repressor to turn the lac operon on

What is one addition for gene expression regulation that some operons have (primarily inducible)?

A CRP-binding site/ CAP site (located in the regulatory region)

What is CAP? Include its function.

CAP (catabolite activator protein) is a stimulatory protein; cAMP binded to CAP (inactive turned active) creates CRP (cyclic AMP responsive) which increases the affinity of RNA polymerase, accelerating transcription

What controls cAMP?

Glucose degrades cAMP preventing the production of CRP

What is the importance of the lac operon?

E. coli use lactose as an energy source when glucose is low; lac operon allows lactose to be broken down for energy

Review: When lactose is absent, is the repressor inactive/active and is the operon on/off? What about when lactose is present, glucose is scarce? Lactose & glucose present?

\-Lactose absent: repressor active, operon off

\-Lactose present/glucose scarce: cAMP levels high, repressor inactive, operon on

\-Lactose & glucose present: cAMP levels low, repressor inactive, operon off

Do eukaryotic cells have operons?

No

Define differential gene expression.

The process where different genes are activated in a cell, giving that cell a specific purpose/function

What are the stages eukaryotic gene expression is regulated at?

1. Chromatin modification
2. Transcription
3. RNA Processing
4. Transport to cytoplasm
5. Degradation of mRNA
6. Protein Processing
7. Degradation of protein
8. Transport to cellular destination

Describe the first stage of where eukaryotic gene expression is regulated at.

Regulation of chromatin modification (controls gene expression based on how unwound the DNA is):

\-DNA unpacking

\-does not alter DNA sequence, but can be passed to future generations of cell (epigenetics)

\-modifies euchromatin/heterochromatin

Euchromatin versus heterochromatin.

euchromatin- unwound (more transcriptionally active); heterochromatin- more compact than euchromatin

What are the three chromatin modifications of the first stage?

Differences between euchromatin and heterochromatin made through:

\-histone acetylation (adding acetyl groups->less compact)

\-histone demethylation

\-histone methylation

How does eukaryotic gene expression affect the second stage?

Transcription affected by DNA methylation

What is DNA methylation?

The addition of methyl groups to certain bases in DNA or promoter (turns off gene expression), is often associated with reduced transcription; can cause long-term inactivation of genes in cellular differentiation

What does DNA methylation affect?

1. Chromatin structure
2. Transcription initiation

How does DNA methylation affect genomic imprinting?

Regulates expression of either maternal or paternal alleles of certain genes during development

Define epigenetics.

The study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself; experiences of previous generations can affect who we are (e.g. smoking, stress, malnourishment)

In order for transcription to occur, what must/must not happen?

Gene available for transcription (no methylation)

Describe the fifth stage of where eukaryotic gene expression is regulated at.

mRNA degradation: the lifespan (longer-\>more protein) of mRNA molecules in the cytoplasm is key in protein synthesis

What is the rate of mRNA degradation controlled by?

\-sequences in the leader and trailer regions

\-length of polyA tail (longer tail->longer lifespan)

\-some RNAs

Why does the prokaryotic mRNA degrade faster?

Lack of cap and tail

What are noncoding RNAs?

RNAs that do not code for a protein; 90% of bacteria chromosome codes for DNA; 1% of human chromosome codes for DNA

What do the other RNAs (that do not code for proteins) encode/what are the other RNAs?

1. Genes for rRNA & tRNA
2. Noncoding RNAs (ncRNAs)
3. MicroRNAs (miRNAs)
4. Small interfering RNAs (siRNAs)

What are microRNAs (miRNAs)?

Small single-stranded RNA molecules that can bind to mRNA;

\-can degrade mRNA or block its translation

\-may regulate expression of at least half of all human genes

What are small interfering RNAs (siRNAs)?

Similar to mRNAs; higher target specificity; made in labs; results in RNA interference (RNAi)

What is RNA interference (RNAi)?

The blocking of gene expression by siRNAs (used in pest resistance)

What are the potential targets of RNAi?

1. Cancer
2. Cardiovascular disease
3. Neurodegenerative disorders
4. Infectious disease
5. Autoimmune disorder
6. Obesity/diabetes

What makes siRNAs difficult to work with?

Delivering it as it needs to go through the cell membrane and nuclear membrane

Describe the sixth & seventh stage of where eukaryotic gene expression is regulated at.

Protein processing and degradation:

\-various types of protein processing, including cleavage and the addition of chemical groups, are subject to control

\-proteasomes

What are proteasomes?

Giant protein complexes that bind protein molecules and degrade them; Folded protein with ubiquitin attached will enter and proteasome, both proteasome and ubiquitin are recycled while the protein become fragments (peptides)

What does ubiquitin signify?

The age of the protein (more ubiquitin-\>protein more likely to be degraded)