Ch 17 Control of Gene Regulation in Eukaryotes – Flashcards

Prokaryotes:
1. bacteria grows in competitive rapid conditions
2. simple needs, and single celled
3. don’t have cell specific gene expression
4. have to respond quickly to changes in their environment
5. organized in operons and transcribed by a single mRNA
6. polycistronic mRNA
7. transcription and translation can occur at the same time

Eukaryotes:
1. have other regulatory needs so they have a different set of regulatory controls
2. are NOT regulated by operons
3. each structural gene has its own promoter and is transcribed independently
4. monocistronic
5. chromatin structure affects gene expression
6. transcription requires unwinding of the DNA form the histone proteins
7. eukaryotic cells have many more transcriptional activators as well as gene repressors (DNA binding proteins)
8. transcription and translation are physically separated so the regulatory mechanisms must differ from those in bacteria

Eukaryotic genes are NOT regulated by operons.

In eukaryotes transcription and translation are separated by the nuclear membrane

Both have DNA binding proteins

transcriptional activators and gene repressors

each mRNA codes for a single polypeptide; therefor each gene codes for a single polypeptide

the mRNA codes for two or more polypeptides; only found in prokaryotes

a gene

DNA tightly wound around the nucleosomes tends to repress transcription.

DNA is associated with the histones and its hold must be released “loosened” in order to have transcription

Efficient transcription requires changes in chromatin structure to make the DNA more accessible to transcription factors, activator proteins, and RNA polymerase

* it is used as a tool to “probe” chromatin structure
* it is not a sequence specific enzyme
* DNA that is tightly packed or would will be resistant to it.
* it is an enzyme that cleaves the DNA if it has access to it

DNA tightly wound around nucleosomes will be resistant to the nuclease since the DNA is protected by the histone proteins

changes in chromatin that make the DNA less tightly associated with the nucleosomes will be seen as an increased sensitivity to degradation by a nuclease

DNA that is most accessible to the enzyme

DNA located ~1000 bp upstream of the promoter of transcriptionally active

8 histone proteins

basic proteins that are positively charges at the physiological pH and bind with the negative charge of the DNA. Thus, holding the DNA tightly together on the nucleosomes.

It is the process of adding an acetyl group. It neutralizes the positive charge on the tails of the histones causing the DNA to become more loosely associated with it. (DNA becomes less tightly packed)

Histones have positively charged tails that interact with the negatively charges phosphates of the DNA

Acetyl group can be transferred to the lysine and arginine amino acids that have the positive charge by acetyl transferases

neutralizes the positive charge so the DNA is more loosely associated with the histone

allowing some transcription factors to bind to the DNA

* a range of proteins whose function is to change the conformation or organization of the chromatin
* some transcription factors or other regulatory proteins alter chromatin structures without affecting histones directly
* in some cases the actual positions of nucleosomes get moved relative to where they were on the DNA

GOAL: if the genes are going to be transcribed they must be in a more euchromatic form / configuration

Nucleosomes that are repositioned to allow binding and initiation of transcription.

CH₃ group

It will almost always occur on a cytosine residue at position 5

repression of transcription; the genes are not expressed

DNA that is available for transcription; genes that are expressed

CpG islands

* this means that a methylated C is usually followed by a G
* methylated C=CG sequences
* generally found in heterochromatic areas
* p represents the phosphodiester bond
* inactive genes have heavily methylated CpG

(there are methylated cytosine on opposite strands)

to block transcription

With fully repressed genes such as those on the inactive X-chromosome of mammalian females.
(ex: insulin promoter in brain tissue)

changes in chromatin by NOT changes in the DNA sequence that are passed on to future generations (affect the germ line)

* changes in the chromatin must be stable through chromosome replication if the epigenetic effect in inheritable
* key mechanism appears to be DNA methylation (CpG islands)

* CpG methylation means that there are methylated cytosine on opposite strands
* after replication the old strand is methylated resulting in hemimethylation
* methyltransferase methylates adds -CH3 to unmethylateted “new” strand
* siRNA, miRNA, and piRNA also play a role in some cases

after replication

1. before replication DNA is fully methylated at CpG dinucleotides
2. during replication the new strands are synthesized without methyl groups
3. after replication each new DNA molecule will have methylation on one stand but not the other; hemimethylated
4. methyl groups attract methylytransferase enzymes, which add methyl groups to the unmethylated strand
5. resulting in fully methylated DNA

When only one strand of DNA is methylated. Occurs in a very short window of time.

A individuals unique chromatin modifications.

* it can be passed on to daughter cells during mitosis
* it can be maintained through DNA replication
* it can affect which genes are active in an individual

General transcription factors assemble and bind to the core promoter that is located immediately upstream of the gene.

enhance the rate of transcription by stimulating (or stabilizing) the basal transcription apparatus

1. One domain of the proteins binds to specific DNA sequence.
2. Another domain can interact with a different component of the transcriptional apparatus to enhance the rate of transcription.

* Requires a coactivator.
!!Transcription will not happen until the transcriptional activator proteins are in place. !!

A protein that can serve to the link the transcription facto to another component of the transcription machinery.

* a regulatory promoter that is upstream of the core promoter
* an enhancer which may be located several thousand bases from the promoter
* (sometimes a sequence can fall in between the 2 categories)

* located upstream of the core promoter
* must be close to the core promoter
* strict requirements on positioning

* function the same as a regulatory promoter
* may be located 1000’s of base pairs away

an example of a transcriptional activator protein

regulates transcription of several yeast genes involved in galactose metabolism

(widely used in molecular biology as a research tool since most other eukaryotes do not have a homologous transcription factor)

The DNA binding site for the GAL₄ protein

Binding activates transcription

It regulates GAL₄ activity.

binds to GAL₄, which prevents binding to UASg so transcription cannot occur.

binds galactose

allosteric change prevents binding of the GAL₈₀-galactose complex to GAL₄, GAL₄ is free to bind to the UASg and transcription initiates

* Galactose is added to the system. (affecter molecule)
* UASg is the upstream activator sequence.
* GAL₄ binds to it and activates transcription.
* GAL₈₀ binds when galactose is not present to repress transcription

* Enhancers can affect transcription even when they are located at a considerable distance from the target gene
* In general, activator protein binds to the enhancer
* The enhancer “loops out” to bring the activator protein closer to the basal transcription apparatus.
* most enhancers could affect any gene within their proximity.

To avoid cross talk across genes.

DNA sequences that function to limit (or isolate) the activity of an enhancer.

Provide a physical barrier to keep the enhancers from enhancing the wrong gene.

some genes need to be expressed in a coordinated manner, but eukaryotic genes are not organized in operons
(ex: heat shock response requires a coordinated response of ~20 genes)

a DNA binding sequence that is the same

A common regulatory DNA sequence shared by genes that respond to a specific condition in the cell (ex: heat shock)

Usually have a short consensus sequence.

(One gene can be activated by a variety of stimuli and have several different response elements)

* a single gene can be activated by several different response elements
* multiple response elements allow a single gene to be activated by different stimuli
* presence of the same response element on different genes allows coordinated expression of a set of genes in response to a common stimulus

mRNA Processing
RNA Stability
RNA Silencing

Alternative splicing of the pre-mRNA to give different variants of the mRNA
(causes the exons to splice together in different ways.)

An RNA binding protein that is synthesized in female Drosophila cells.

It binds to the tra (transformer) pre-mRNA and affects splicing.

a protein that is coded for in both sexes of Drosophila but there is a difference in the splicing of the pre-mRNA

it is required for female development; males have a nonfunctional form of this protein

(males don’t have a functional tra protein not because the gene wasn’t transcribed but because it was spiced in a way that lead to a non-functional protein.

* In the absence of sxl protein (males), splicing of the tra mRNA occurs at a different place.
* In females, the sxl protein binds to the pre-mRNA to prevent splicing. The result is splicing at an alternative site.
* The alternative splicing in females leads to a tra mRNA that codes for a functional protein.

* eukaryotic mRNA is, in general, more long lived than prokaryotic mRNA
* some eukaryotic mRNAs are stable for hours, days or even months
* can result in very large differences in the amount of protein made
* (ex: red blood cells)

a mechanism for silencing gene expression my targeting the RNA for early degradation rather than allowing it to degrade at its own rate

(possibly as many as 30% of genes utilize RNA silencing)

* processes that occur during protein synthesis or after the protein has been synthesized
* there are factors that can bind the mRNA in the 5′ end UTR (untranslated region), that can affect the rate of translation
* rare but, some factors bind to the 3′ UTR to affect translation

* many proteins are initially made in an inactive state

* activation requires addition processes such as removal os some amino acids, as well as chemical modification (methylation, acetylation, phosphorylation etc)