xii. gene regulation. - overview: all cells in an organism contain the same genetic information; the...

XII. Gene Regulation

- Overview:

All cells in an organism contain the same genetic information; the key to tissue specialization is gene regulation – reading some genes in some cells and other genes in other cells.

Also, organisms can respond to their environment at a genetic level, so there must be a way for the environment to stimulate or repress the action of certain genes.

And changes occur through time, creating developmental changes. We will look at how gene expression is regulated in these cases.

- Overview:

-Some Terminology:

- some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.

- Overview:

-Some Terminology:

- some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.- some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present.

- Overview:

-Some Terminology:

- some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.- some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present.- Constitutive genes are on all the time.


A. The lac Operon in E. coli



- When lactose is present, E. coli produce three enzymes involved in lactose metabolism. Lactose is broken into glucose and galactose, and galactose is modified into glucose, too. Glucose is then metabolized in aerobic respiration pathways to harvest energy (ATP). When lactose is absent, E. coli does not make these enzymes and saves energy and amino acids. How do they KNOW? : )



As you remember, an “operon” was a region of genes that are regulated as a unit – it typically encodes > 1 protein involved in a particular metabolic pathway.



Lac Y - permease – increases absorption of lactose




Lac Z – B-galactosidase – cleaves lactose into glucose and galactose




Lac Z – B-galactosidase – cleaves lactose into glucose and galactose

Lac A – transacetylase – may code for enzymes that detoxify waste production of digestion.


A.The lac Operon in E. coli

1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription.

Repressor Gene

Repressor RNA Poly

Operator

Promoter




LACTOSE




LACTOSE

The binding of lactose changes the shape of the repressor (allosteric reaction) and it can’t bind to the operator.



Mutant analyses confirmed these results:



Mutant analyses confirmed these results:

Curiously, there are only about 10 repressor molecules in each cell and they were not actually isolated and identified for 6 years (Gilbert).



But it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW?



But it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW?

This involves a repressible pathway.



Within the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter.

, lactose present



Within the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter. So, the binding of CAP stimulates transcription.

, lactose present



When Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off.

, lactose present

CAP

REPRESSOR



When Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off.

So, the lac operon is regulated first by the presence/absence of glucose; the needed nutrient…and then by the presence of lactose, which could be metabolized to produce glucose if necessary.



B. The trp Operon in E. coli




Tryptophan is an amino acid that can be synthesized by tryptophan synthetase. This gene and its partners are only ON if tryptophan is absent. The presence of tryptophan represses the production of these enzymes (repressible system).


If trp is absent, the repressor can’t bind to the operator… transcription proceeds..


If trp is present, it binds to the repressor, changing the repressor’s shape so that it can now bind to the operator and inhibit RNA poly binding.


Actually, when trp is present,

Secondary Regulation


ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”…



Two hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription.




Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence.





If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms…





If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms…

If low trp, then ribosome stalls; region 3 bind to 2, no termination loop forms, and transcription of the genes proceeds… Translation of the genes begins at start codons downstream…





C. Regulation in Eukaryotes





- higher levels of packaging, intron-exon structure, and the need for tissue specialization makes regulation in eukaryotes far more complex than responding to environmental cues.





- higher levels of packaging, intron-exon structure, and the need for tissue specialization makes regulation in eukaryotes far more complex that responding to environmental cues.

1.Histone Regulation

- Core DNA, bound to histones, is OFF. Only “linker DNA”, between histones, is even accessible to RNA polymerases. So, binding DNA to histones is the first way to shut it off.


1. Histone Regulation

- Three ways to reveal DNA“chromatin remodeling”




2. Methylation

- highly repetitive sequences - imprinted genes - Barr bodies




2. Methylation

- highly repetitive sequences - imprinted genes - Barr bodies

Some proteins bind to the methylated cytosines, and may either recruit repressors or interrupt transcription factor binding.



2. Methylation

3. Promoters

- Several consensus sequences (TATA, CAAT, GGGCGG) appear in combination in nearly all promoters and are required for basal levels of transcription



2. Methylation

3. Promoters

4. Enhancers/Silencers

Cis-acting elements on the same chromosome, which regulate a neighboring gene.

They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or after



2. Methylation

3. Promoters


Cis-acting elements on the same chromosome, which regulate a neighboring gene.

They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or after

They are not gene specific – they will enhance their neighbor

Silencers tend to reduce binding of the polymerase to the promoter.



2. Methylation

3. Promoters


These are the transcription factors that bind to enhancer and silencer regions of the human metallothionien IIA gene promoter region!!- What does having all these modifiers allow for?

Different proteins can silence or enhance DNA Polymerase II binding. This may involve the formation of a pre-initiation complex” of proteins that allow the Poly II to bind, and can even involve sequences far from the promoter that loop – and influence RNA Poly II activity.



5. Transcription Factors

- These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.





HTH = “helix-turn-helix”

One class of important HTH TF’s contain specific sequences of AA’s called a homeodomain. This is encoded by a 180 bp region in it’s gene called a homeobox. These homeotic genes/proteins are conserved across all eukaryotes and are critical to basic animal development.





“Zinc-Finger”: Zinc binds to two cysteine and two histidine AA’s. The sequence between formsA loop or “finger”, and the specific AA sequenceBinds specific DNA sequences…




We didn’t really know what they did in vivo. Biochemists have linked other proteins to them, however, making Zinc-finger nucleases that cut DNA at specific sequences.




But Feb 18, 2015, Najafabadi et al. found this:

- “Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors” (> 700 proteins)

- “C2H2-ZF proteins recognize more motifs than all other human transcription factors combined.” (Highly variable, tough to study)

- “C2H2-ZF proteins bind specific endogenous retroelements (EREs), ranging from currently active to ancient families. The majority of C2H2-ZF proteins, also show widespread binding to regulatory regions, indicating that the human genome contains an extensive and largely unstudied adaptive C2H2-ZF regulatory network that targets a diverse range of genes and pathways.”

- They stabilized Endogenous Retroviral Elements, and evolved to regulate other genes, as well.

Najafabadi et al. 2015 Science DailyREAD THIS

http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3128.html

http://www.sciencedaily.com/releases/2015/02/150220133346.htm

http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3128.html





bZIP=“basic leucine zipper”: leucine AA’s inDifferent chains dimerize and the leucines “zip”

The other alpha-helices bind specific DNA sequences





- Then, the TF’s have other binding sites forProteins (like basal transcription factors) orOther chemicals (like hormones)



6. Alternate Splicing Pathways

- Many proteins can be made from the same gene, by splicing the m-RNA differently. Humans have 20-30K genes, but several 100,000 proteins!

A calcium regulator in the thyroid

A hormone made in the brain


6. Alternate Splicing Pathways

7. Controlling m-RNA stability

Existing tubulin units interact with a new tubulin strand and translation stalls, releasing RNAse that cleave the m-RNA.

So tubulin is only made when free tubulin units are not present.



8. RNA Silencing

- Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression.



8. RNA Silencing/Interference

- si-RNA (small interfering RNA):Viral or retrotransopon origin

- mi-RNA (micro-RNA):Produced by intronic, sequences,

or different genes in genome. Have stem-loop structure.




THEY BOTH are attacked by DICER protein, which cuts them into short ds-RNA molecules.

These complex with RNA-induced Silencing Complex proteins (RISC) that denature the RNA and degrade the sense strand.

What is left is a strand that is complementary to a specific m-RNA molecule.




If the ss-RNA is exactly complementary to a m-RNA, RISC cuts the m-RNA into fragments (turning protein synthesis OFF).

Or, if not exactly complementary, then the RISC complex stays attached, interrupting ribosome binding and translation.




OR!

The ds-RNA gets complexed with RNA-induced initiation of transcription silencing complex (RITS). These denature the RNA, creating ss-RNA that binds to DNA promoters or large regions of DNA.

This binding attracts chromatin remodeling proteins that methylates the histones, causing it to coil into herochromatin (Turning Genes OFF).

The process of Gene Activity – in terms of a Gene Making a Functional Protein – can be regulated at every step of the process, from:

Gene availability and chromatin structure

Transcription

Transcript Processing

Translation

Post-translational Modification

Variation in patterns of regulation lead to differences in expression between cells, and cell specialization.

xii. gene regulation. - overview: all cells in an organism contain the same genetic information; the...

Documents

gene regulationa

lac operon

enzymatic genes

region of genes

gene expression

gene regulation overview

constitutive genes

inducible system