Business of the day

Today: Ch. 18 Regulation of Gene Expression Chapter 18 homework due next Thursday at 9 am Homework Ch 13 and 14 due today at 9 am, Ch 15 due Tuesday at 9 am. Exam: one week from today here at class time; Ch. 12-18 Next help session this afternoon at 5 pm in Blake Special help session on Sunday at 5 pm in 214 Foster come with questions

Chapter 18: Regulation of Gene Expression

Genes and metabolic pathways need to be turned on and off at appropriate times. Otherwise, organisms would be making unnecessary products and wasting resources. Regulation of gene expression refers specifically to the cellular mechanisms that turn genes on and off or control the appearance and longevity of gene products (like proteins). Metabolic control: This is somewhat different. It involves turning gene products (mostly enzymes) on and off.

Regulation of Bacterial Gene Expression

E. coli in the gut have no control over the hosts diet (and they eat what the host eats), so they may have to manufacture certain nutrients themselves (e.g., the amino acid tryptophan--Trp)

There are two ways to control the production of Trp 1. Metabolic control: Control the metabolic pathway that makes Trp (control enzymes) Control of gene expression: Control transcription of enzymes that help build Trp

2.

1. Metabolic control turns enzymes (and thus enzyme pathways) on or off quickly

The Tryptophan anabolic pathway requires several steps, each mediated by an enzyme Trp can control its own production by feedback inhibition If there is lots of Trp available to a bacterium, it will inhibit the anabolic pathway that makes Trp by interfering with enzyme 1.

Metabolic control is based on feedback inhibition (negative feedback)

From Ch. 6: The end product inhibits an enzymatic pathway

An enzymes allosteric site

Positive Feedback: sometimes binding to an allosteric site increases enzyme activity and increases the rate of production (we will see this with the Lac Operon later in this chapter).

2. In addition to metabolic control, regulation of gene expression can be used to control the production of Trp Bacteria can turn on (or shut down) the genes that produce enzymes. This is almost always done by controlling transcription.

Gene Regulation: Controlling transcription

In bacteria, genes are often located in operons (Jacob & Monod 1961) Operons consist of several genes controlled by one promoter. Inhibiting (or stimulating) that promoter slows (or speeds up) mRNA production

The operator determines whether RNA polymerase can proceed

start and stop codons cause each gene to be translated separately into polypeptides

Trp Operon Negative Control: Trp shuts down trp operon

Tryptophan is an amino acid produced with the help of a series of enzymes. These enzymes are encoded by genes in the Tryptophan Operon. Transcription of the genes in the Trp operon is turned off by a protein called a repressor, which sits on the Trp operator and blocks RNA polymerase. The Repressor is a protein encoded by DNA elsewhere in the genome by a regulatory gene. It is a factor specific to the Trp operon. The repressor is an allosteric protein that has active and inactive conformations. It is inactive at synthesis and becomes activated by joining with tryptophan. Corepressoractivates the repressor (in this case the corepressor is Tryptophan)

Negative Control continued

The repressor shuts down transcription with help of the co-repressor Trp. This is an example of negative feedback (or negative control)because the end-product (Trp) shuts down its own production.

Trp operon is Repressible. Normally its turned on, but its inhibited when Trp is abundant.

TrpR gene (regulatory) Trp operator

Lac Operon: An inducible operon

Glucose

Galactose

Bond hydrolyzed by -galactosidase

Background: Jacob & Monod (1961) Lactose = Milk sugar. It is a disaccharide of glucose and galactose When you drink milk, E. coli in your gut digest lactose to obtain glucose (and galactose) for energy (ATP) production

Lac operon structure

Three structural genes comprise this operon 1. -galactosidase: hydrolyzes the lactose dimer into the 2 sugars 2. Permease: A membrane protein that moves lactose into the cell 3. Transacetylase its purpose is not fully understood
lac repressor gene

Lac operon repressor

Default: The lac operon is turned on by defaultbut, a repressor protein is produced in active form, so it binds to the operator and shuts off the operon. The repressor binds without a corepressor. So, the lac operon is generally turned off.

Lac operon repressor, continued

When present, lactose binds to the repressor (technically allolactose, a lactose isomer, binds to the repressor) This deactivates the repressor, so that it cant bind to the operator Thus, transcription is induced by lactose (allolactose)

Lac operon, penultimate summary

The Lac Operon is an inducible operon Induced (turned on) when lactose is abundant The Trp Operon is a repressible operon Repressed (turned off) when Trp is abundant Inducible operons generally involve catabolism The operon is activated to break down a molecule Repressible operons involve anabolism The operon is activated to build a molecule Both are examples of negative gene regulation or negative control of genes because the operon is shut down by the active form of the repressor

Lac Operon: Also has positive gene regulation

Lac operon requires more than lactose to be fully activated. To be completely efficient, glucose must be depleted. Why? Besides the operator, which can be blocked by a repressor to turn transcription off, the Lac operon promoter has a CRP binding site, which controls the overall rate of transcription when the operon is on. CRP = cyclic AMP Receptor Protein: a regulatory protein that enhances the binding of RNA polymerase, thus stimulating transcription. This is positive gene regulation because the operon is stimulated by an activator molecule. cAMP

activated by CRP attachment

Lac Operon
The Lac Operon is turned on (induced) when Lactose (allolactose) deactivates its repressor The CRP is activated by cAMP. When activated, CRP binds to DNA at the CRP binding site next to the promoter and enhances RNA polymerase binding, thus improving the efficiency of transcription. Thus, the Lac Operon is most efficient when glucose concentration is low and cAMP concentration is high.

Whats with cAMP?

cAMP = cyclic adenosine monophosphate. It is the energetically depleted relative of ATP. High cAMP concentration signals low glucose content. Why? Why is low glucose content important to the lac operon? When glucose is low, ATP gets used up and AMP increases in concentration. The bacterium needs to catabolized lactose to glucose for respiration to produce more ATP from the AMP.

Lac operon, final summary

When no lactose, there is no lac operon activation (the repressor is active) Cells wont waste ATP, etc., making enzymes to breakdown lactose when there is no lactose. When lactose and ATP are abundant, there is slight activation of the operon because the repressor is deactivated and RNA polymerase works at a low level

Lac operon, final summary continued

When there is lactose and lots of cAMP, that means ATP is low, and the lac operon works at peak efficiency. Lactose binds and inactivates the repressor cAMP activates CRP which binds to the DNA. RNA polymerase then jumps on the promoter like a duck on a June bug.

The on-off switch & volume analogythink rheostat

Structural vs regulatory genes

Structural genes code for enzymes and other proteins Regulatory genes include the genes that produce products like regulatory proteins that bind to operators, cAMP receptor protein, and transcription and translation factors. These control the expression of structural genes.

Regulation of eukaryotic gene expression

Gene Expression: Refers to the timing and circumstances in which genes do their work. Obviously, all genes cant be turned on all the time. Control of gene expression depends largely on genome organization. Multicellular eukaryotes have the special problem of cellular differentiation: specialized cells (e.g., liver, heart, muscle), need to turn their genes on and off at different times. Some genes are never expressed! When can gene expression be controlled? In chromatin (packing & unpacking) During transcription During and after mRNA processing During and after translation.

Chromatin: Replication & Transcription

When tightly packed, DNA in chromatin cannot be not replicated or transcribed

Chromatin packing loosens to allow replication & transcription -Euchromatin: Loosely packed (ready for action) -Heterochromatin: Never unpacked, except for interphase replication

Introduction to genome organization

Understanding genome organization is the first step to understanding gene control. -Expression of genes is partly a function of how they are packed and ordered. Chromatin Packing: -Chromatin: Complexes of DNA and protein -Histones: Proteins playing a key role in DNA packing -Nucleosomes: Beads of histones wrapped with DNA -Chromatin fiber: Coils of nucleosomes -Looped domains: chromatin fiber attached to a nonhistone scaffold -Compacted chromatin folds = visible chromatids

More common mechanisms for controlling gene expression

Because of cellular differentiation, only 3-5% of genes need be expressed in a given cell How is this expression most commonly controlled? Methods of contol include: Histone acetylation (--COCH3): Loosens histone grip on key genes DNA methylation (--CH3): Deactivates unnecessary genes

Control of Transcription

Most gene expression is controlled at the transcription stage.

Control of Transcription: Control Elements

Transcription Factors: Proteins that bind to DNA to help activate or deactivate transcription Control Elements: DNA sites that bind transcription factors Enhancers: Distal control elementsDNA sequences far from the gene Proximal control elements: DNA sequences close to promoter.

Control of Transcription: Factors

Transcription factors: proteins that affect timing and expression of gene by binding to the DNAs elements Every protein that facilitates or inhibits transcription is a factor. Factors recognize elements or other factors.

Control of Transcription: Activators

Activators are factors that bind to Enhancers (i.e., at sites distant from the promoter).

How do activator/enhancers help when they are located far away?

Bending of DNA places these distant complexes at the promoter.

Control of Transcription: Other issues

Are there repressors in eukaryotes like the ones in prokaryote operons? Yes, silencers can bind to control elements and block activators and other transcription factors, but eukaryotic genes are turned off by default, so mostly they need to be turned on, not off.

prokaryotic repressor

Control of Transcription How do factors attach to DNA

Proteins consist of domains with specific functions. Transcription factors have domains that allow them to bind to DNA. All transcription factors share similar DNA-binding domains A generic protein

Control of Translation-mRNA processing

After transcription: pre-mRNA must be converted to mRNA in the nucleus. Control can consist of: 1. Failure or slowing of this processing 2. Alternative splicing of introns to produce different mRNAs from same premRNA.

Movie

Control of Translation: mRNA Degradation

In the cytoplasm: mRNA tends to be a fragile molecule that breaks down easily The longer it survives, the more protein it codes Example: Hemoglobin mRNA is long-livedthis increases production of hemoglobin in red blood cells.

RNA Interference: a mechanism for controlling mRNA expression. Small interfering RNAs (siRNA) or micro RNA (miRNA) are the key in mRNA degradation
1 The microRNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 2 An enzyme called Dicer moves along the doublestranded RNA, cutting it into shorter segments. 3 One strand of each short doublestranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 4 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. 5 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation.

Chromatin changes

Transcription

RNA processing

mRNA degradation

Translation

Protein complex

Protein processing and degradation

Dicer Degradation of mRNA OR miRNA Target mRNA

Hydrogen bond

Blockage of translation

Control of translation: Stopping the act

Translation is stopped mostly at initiation Two general ways: 1. Regulatory proteins bind to mRNA initiation site 2. Translation initiation factors are inhibited, e.g., by phosphorylation.

Post-translational Control
This occurs in various ways: 1. Processing of peptides is slowed, stopped, or speeded up. e.g., insulin needs to be cleaved to work e.g., hemoglobin needs its four parts assembled e.g., membrane proteins often need sugars to be attached
2. Selective degradation -analogous to mRNA degradation -ubiquitin attaches to proteins & proteasomes gobble them up

Control of eukaryotic gene expression Summary

packaging number of gene copies transcriptional control (factors and elements) post-transcriptional modification of mRNA

rate of mRNA degradation

control of translation rate of protein degradation

Genome organization: Prokaryotes vs. Eukaryotes

Prokaryotes: Most DNA consists of genes that code for mRNA, tRNA, & rRNA Genes are grouped into operons Eukaryotes: 97% of DNA is not genes, does NOT code for mRNA, tRNA, or rRNA Repetitive DNA: non-coding, repeating sequences Each gene controlled by a separate promoter--no operons known