Midterm 3 Flashcards
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Basal level
Neither activated or repressed
Different stages of gene regulation
Transcriptional and translational
Repressed
Regulation occurs through negative control
Activated
Positive control
Components of regulation
DNA regulatory sequences, regulatory proteins, small effector molecules
Cis elements
DNA regulatory seq
Trans elements
Proteins that bind to DNA regulatory seq and affect transcription of one or more genes
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
Allosteric activation
Activators interact with the closed complex and induce a conformational change that leads to open complex and initiates transcription
DNA looping
Sometimes proteins are needed to help DNA loop around and have the activator bind to the site it needs to activate
Operon
A cluster of genes that are under the transcriptional control of a single promoter in prokaryotes
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
Lac repressor is a…
Tetramer!
What happens when lactose is absent?
The lac repressor binds to the operator and inhibits transcription
What happens when lactose is present?
Allolactose acts as an inducer and binds to the repressor making it inactive so transcription can begin again
How does CAP activate the lac operon?
It recruits RNA polymerase
Function of cAMP
It binds to the CAP which will then bind to the CAP site near the lac promoter and hence increase transcription
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
X-gal
It is a substrate for beta galactosidase but not an inducer
What happens when the repressor is supplied in trans
The lac genes continue to be expressed constitutively like the mutant
What does the trp operon code for?
Codes for the enzymes required to make amino acid tryptophan
How is the tryp operon regulated
Through repression and attenuation
Attenuation
Early termination of transcription
What happens when tryptophan levels are low?
The repressor cannot bind to the operator site and the genes are transcribed
Other types of transcriptional regulation in prokaryotes
Different sigma factors, allosteric activators, some repressors hold RNA poly at the promoter, antiactivation
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
Transcriptional regulation in eukaryotes
Nucleosomes and modifiers of chromatin structure do most of regulation. There are many more regulators and extensive regulatory sequences
Positive regulation in eukaryotes
Activators and enhancers
Negative regulation in eukaryotes
Repressors and modification of histones and DNA
The two functions of activators
They have DNA binding and activating functions
Domain swap experiment
The activation domain will work as long as it is connected to another DNA binding domain
Eukaryotic binding partners
Homodimers, heterodimers, monomers
Common domains in eukaryotic regulators
Homeodomain, zinc finger, leucine zipper, helix loop helix, HMG (high mobility group)
Leucine zipper
Always dimeric because it has a dimerization domain and a DNA binding domain. It grasps the DNA in a scissors grip
HMG
The high mobility group interacts with the minor groove and alters the DNA conformation
What do activators recruit in eukaryotes?
Transcriptional machinery and nucleosome modifiers
The mediator complex
It mediates the recruitment of RNA pol ll by interacting with the tail of RNA pol ll.
TFllD
It is the first factor to bind to the promoter region and recruits all the other factors
The ground state of eukaryotic genes
Repressed! Without activators, genes are not expressed
Example of a histone modifier
Histone acetyltransferases (HAT)
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
The three types of modifications
Methylation, acetylation, and phosphorylation
Insulators
They limit the spread of chromatin modification so that enhancers can’t turn on too many genes, mo specific
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
Where does methylation usually occur?
On cytosine bases having a guanine after them
Another thing that methylation does to genes
It causes proteins to bind to the DNA which blocks the transcriptional machinery from binding
Imprinting
When individuals only express their maternal or paternal allele but not both. This happens because of different states of DNA methylation
Epigenetics
The inheritance of variation above and beyond changes in DNA sequence, which is also self perpetuating and reversible
Synergistically
When two or more factors work together to make an effect greater than the sum of their individual effects
How many activators is the HO gene controlled by?
Two! It needs both to turn on
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
Combinatorial regulation
More than one regulator a required to activate each gene. This allows less factors to do more function than individual factors
How as different cell types created?
They are generated by the presence of different regulatory factors
How can hormones affect gene expression?
They can ass through the cell membrane because they are nonpolar and then bind to glucocorticoid receptors
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
Cis regulation in bacteria
Riboswitches and attenuation
Riboswitches
They are found in the 5’ UTR and they function through changes in RNA secondary structure
What signal do riboswitches respond to ?
They respond to metabolites
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
Regulatory RNA in eukaryotes
RNA interference, and x inactivation
RNA interference
Inhibits gene expression using noncoding RNA molecules with complementary sequences to target genes
Where/how are siRNA made?
They are produced in the cell from dsRNA precursors or made artificially
How are miRNA made?
They are derived from precursor RNA encoded in the genome
RISC
RNA induced silencing complex.
What does drosha do?
It cleaves pri-miRNA into pre-miRNA. It is a RNAse III enzyme which is specific for ds RNA
Argonaute
It is the central component of RISC that turns dsRNA into a single strand and then forms a complex with that single strand
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
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
Xist
It is a long noncoding RNA that inactivates a single X chromosome in female mammals
How do cells regulate alternative splicing?
Activators can bind to splicing enhancers, and repressors can bind to splicing silencers
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
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
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
What type of gel are the fragments for sequencing separated on?
A polyacrylamide gel and the bands a read by autoradiography
What can we sequence?
We can sequence recombinant vectors, PCR products and whole genomes
How can you sequence whole genome libraries?
Through shotgun sequencing or paired end sequencing
Contig
Larger contiguous sequences made from short random shotgun seq put together. Overlapping regions allow researchers to determine their order along the chromosome
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
The two common DNA libraries
Genomic and complementary DNA
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
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
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.
Nonsense mediated mRNA decay
When a premature stop codon is reached,
Non-stop mediated decay
The entire polypeptide chain is made, and then everything is degraded
No-go-mediated mRNA decay
When a ribosome is stalled
DNA sequencing
It is used to determine the base sequence of DNA. There is Sanger sequencing and Second generation sequencing
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
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
Two common types of DNA libraries
Genomic library and cDNA library
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
qRT PCR
Quantitative reverse transcription PCR. It allows one to accurately measure products generated during each cycle of PCR
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
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
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
