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Different stages of gene regulation

Transcriptional and translational

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Basal level

Neither activated or repressed

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Repressed

Regulation occurs through negative control

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Activated

Positive control

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Components of regulation

DNA regulatory sequences, regulatory proteins, small effector molecules

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Cis elements

DNA regulatory seq

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Trans elements

Proteins that bind to DNA regulatory seq and affect transcription of one or more genes

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What do small effector molecules do?

They bind to regulatory proteins and cause conformational change in order to determine whether the proteins can bind to DNA or not

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Allosteric activation

Activators interact with the closed complex and induce a conformational change that leads to open complex and initiates transcription

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DNA looping

Sometimes proteins are needed to help DNA loop around and have the activator bind to the site it needs to activate

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Operon

A cluster of genes that are under the transcriptional control of a single promoter in prokaryotes

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The two regulatory sites in the lac operon

CAP site which is in charge of positive control and the operator which is in charge of negative control

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Lac repressor is a...

Tetramer!

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What happens when lactose is absent?

The lac repressor binds to the operator and inhibits transcription

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What happens when lactose is present?

Allolactose acts as an inducer and binds to the repressor making it inactive so transcription can begin again

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How does CAP activate the lac operon?

It recruits RNA polymerase

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Function of cAMP

It binds to the CAP which will then bind to the CAP site near the lac promoter and hence increase transcription

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Function of glucose

It inhibits the production of cAMP and therefore prevents the binding of CAP to DNA and inhibits transcription of the lac operon

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X-gal

It is a substrate for beta galactosidase but not an inducer

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What happens when the repressor is supplied in trans

The lac genes continue to be expressed constitutively like the mutant

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What does the trp operon code for?

Codes for the enzymes required to make amino acid tryptophan

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How is the tryp operon regulated

Through repression and attenuation

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Attenuation

Early termination of transcription

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What happens when tryptophan levels are low?

The repressor cannot bind to the operator site and the genes are transcribed

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Other types of transcriptional regulation in prokaryotes

Different sigma factors, allosteric activators, some repressors hold RNA poly at the promoter, antiactivation

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What happens when tryptophan levels are high?

Tryptophan binds to the trp repressor and this enables it to bind to the operator site which inhibits transcription

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Transcriptional regulation in eukaryotes

Nucleosomes and modifiers of chromatin structure do most of regulation. There are many more regulators and extensive regulatory sequences

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Positive regulation in eukaryotes

Activators and enhancers

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Negative regulation in eukaryotes

Repressors and modification of histones and DNA

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The two functions of activators

They have DNA binding and activating functions

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Domain swap experiment

The activation domain will work as long as it is connected to another DNA binding domain

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Eukaryotic binding partners

Homodimers, heterodimers, monomers

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Common domains in eukaryotic regulators

Homeodomain, zinc finger, leucine zipper, helix loop helix, HMG (high mobility group)

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Leucine zipper

Always dimeric because it has a dimerization domain and a DNA binding domain. It grasps the DNA in a scissors grip

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HMG

The high mobility group interacts with the minor groove and alters the DNA conformation

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What do activators recruit in eukaryotes?

Transcriptional machinery and nucleosome modifiers

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The mediator complex

It mediates the recruitment of RNA pol ll by interacting with the tail of RNA pol ll.

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TFllD

It is the first factor to bind to the promoter region and recruits all the other factors

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The ground state of eukaryotic genes

Repressed! Without activators, genes are not expressed

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Example of a histone modifier

Histone acetyltransferases (HAT)

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The two ways chromatin structure can be altered

Through acetylation which makes the histones loose. Or through remodeling the nucleosome s which may expose the promoter. Both allow the transcriptional machinery to access the promoter

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The three types of modifications

Methylation, acetylation, and phosphorylation

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Insulators

They limit the spread of chromatin modification so that enhancers can't turn on too many genes, mo specific

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The possible mechanisms of eukaryotic repressors

Competitive binding, masking of the activation surface, and direct interaction with the transcription factors so they can't be activated. They can also recruit histone modifiers to take off acetylations or methylate things

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Where does methylation usually occur?

On cytosine bases having a guanine after them

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Another thing that methylation does to genes

It causes proteins to bind to the DNA which blocks the transcriptional machinery from binding

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Imprinting

When individuals only express their maternal or paternal allele but not both. This happens because of different states of DNA methylation

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Epigenetics

The inheritance of variation above and beyond changes in DNA sequence, which is also self perpetuating and reversible

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Synergistically

When two or more factors work together to make an effect greater than the sum of their individual effects

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How many activators is the HO gene controlled by?

Two! It needs both to turn on

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What does yeast use to sense glucose in the cell?

It uses two proteins: MigI and TupI. When these two proteins forms a complex, they bind to DNA and inhibit transcription

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Combinatorial regulation

More than one regulator a required to activate each gene. This allows less factors to do more function than individual factors

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How as different cell types created?

They are generated by the presence of different regulatory factors

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How can hormones affect gene expression?

They can ass through the cell membrane because they are nonpolar and then bind to glucocorticoid receptors

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Trans regulation in bacteria

sRNAs can either bind to the RBS to hide it or they can unmask the RBS by binding to the sequence that was hiding it

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Cis regulation in bacteria

Riboswitches and attenuation

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Riboswitches

They are found in the 5' UTR and they function through changes in RNA secondary structure

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What signal do riboswitches respond to ?

They respond to metabolites

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In the trp operon, what happens when there is low tryptophan?

The leader peptide calls for multiple tryptophans, therefore if the cell is low, the ribosome will stall at the tryptophan codons in the leader sequence

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Regulatory RNA in eukaryotes

RNA interference, and x inactivation

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RNA interference

Inhibits gene expression using noncoding RNA molecules with complementary sequences to target genes

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Where/how are siRNA made?

They are produced in the cell from dsRNA precursors or made artificially

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How are miRNA made?

They are derived from precursor RNA encoded in the genome

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RISC

RNA induced silencing complex.

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What does drosha do?

It cleaves pri-miRNA into pre-miRNA. It is a RNAse III enzyme which is specific for ds RNA

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Argonaute

It is the central component of RISC that turns dsRNA into a single strand and then forms a complex with that single strand

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Some key differences between miRNA and siRNA

MiRNA is only partially complementary to target genes, while siRNA is completely complementary. MiRNA is naturally produced in the cell, while usually siRNA is produced artificially to suppress specific genes

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X inactivation

It occurs at a very early stage in embryonic development where the X chromosome is inactivated. It happens because it gives equal levels of expression of x linked genes in male and female cells

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Xist

It is a long noncoding RNA that inactivates a single X chromosome in female mammals

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How do cells regulate alternative splicing?

Activators can bind to splicing enhancers, and repressors can bind to splicing silencers

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How does alternative splicing lead to pluripotency?

Depending on how FOXP1 is spliced, will determine whether pluripotency genes are created or differentiated genes are created

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Regulation of ferritin translation by iron

Ferritin mRNA has a iron regulatory element that prevents translation when there's no iron and enables translation when iron is present

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What does the cell do with defective mRNAs?

It degrades them in eukaryotes and ssrA rescues the ribosomes that translate these broken mRNA in prokaryotes

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What type of gel are the fragments for sequencing separated on?

A polyacrylamide gel and the bands a read by autoradiography

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What can we sequence?

We can sequence recombinant vectors, PCR products and whole genomes

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How can you sequence whole genome libraries?

Through shotgun sequencing or paired end sequencing

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Contig

Larger contiguous sequences made from short random shotgun seq put together. Overlapping regions allow researchers to determine their order along the chromosome

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Second generation sequencing

DNA templates are put on flow cell surface and the machine can run billions of reactions per time. You only need a small amount of DNA template and it is so much faster

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The two common DNA libraries

Genomic and complementary DNA

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The difference between genomic and cDNA libraries

Genome includes everything including introns while cDNA only has the genes for the proteins being expressed in the cell

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qRT PCR

Quantitative reverse transcription PCR. It allows you to accurately quantitate the amount of DNA you are amplifying using a fluorescent dye. You can measure the products generated through PCR during each cycle

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SsrA

It is part mRNA and part tRNA. It goes inside a stalled ribosome and starts creating a tagged peptide chain from its own sequence that will signal the cell to degrade it.

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Nonsense mediated mRNA decay

When a premature stop codon is reached,

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Non-stop mediated decay

The entire polypeptide chain is made, and then everything is degraded

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No-go-mediated mRNA decay

When a ribosome is stalled

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DNA sequencing

It is used to determine the base sequence of DNA. There is Sanger sequencing and Second generation sequencing

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Sanger sequencing

dideoxynucleoside triphosphates are added and they will prematurely terminate creating many different size fragments of DNA. Each ddNTP is radiolabeled with a different color and added to different tubes so we can see which letter is at which position

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Second generations sequencing

DNA templates are immobilized on a flow cell surface and a machine runs billions of reactions per time. It only needs a small amount of DNA template and is very fast

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Two common types of DNA libraries

Genomic library and cDNA library

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The difference between genomic and cDNA library

Genomic includes everything like introns while cDNA only has the genes that code for proteins being expressed in that cell

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qRT PCR

Quantitative reverse transcription PCR. It allows one to accurately measure products generated during each cycle of PCR

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DNA microarray

mRNA from cells is taken to make cDNA are added to wells. Each well has a short seq of known genes so we can figure out which genes are being expressed or down regulated in the cell

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EMSA

Electrophoretic mobility shift assay can help us determine which proteins bind to a specific DNA sequence. The bound DNA will move more slowly in gel than unbound DNA

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ChIP

Proteins are crosslinked with DNA and an antibody is added targeting the protein of interest. We immunoprecipitate for that protein and then amplify the sequence it was bound to through PCR