28: Regulation of Gene expression Flashcards
7 processes that regulate ‘gene expression’ (be clear in defining what expression is, usually its measured as protein but is sometimes RNA)
Transcription post transcriptional processing mRNA degradation translation post translational processing protein degradation protein targeting and transport
describe prokaryotic gene and RNA structure
gene has regulatory sequences, promoter, and trancripted sequence ending with termination signal sequence.
RNA has 5’ UTR (ribosomal binding site) and 3’ UTR. the translated part (ORF) of the RNA is between 5’ UTR and 3’ UTR
does not have introns.
define housekeeping genes, constitutively, and induction/repression
housekeeping genes: genes are expressed at a constant level in every cell of a species or organism
constitutively: gene shows unvarying levels of expression
any gene which shows increased/decreased levels of expression with some stimulus is induced/repressed. products that increases/decrease under particular circumstances are inducible/repressible. process of inducing/decreasing expression is induction/repression
factors that determine how well RNA polymerase binds the promoter in prokaryotes
- regulatory DNA sequences; -35 and -10 regions are promoter regions that vary and affect binding of polymerase
- regulatory proteins and RNAs: sigma factor has specificity, transcription factors, activators, repressors, riboswitches
- how the genes are arranged: operon structure will affect polymerase binding and which genes are exposed as they are transcribed as a group of genes (only in prokaryotes)
two types of regulation of transcription using protein factors
negative: uses repressor molecules that bind to the operator and block RNA polymerase from associating. signal molecules can bind the repressor and cause it to block or unblock. Prokaryotes use this regulation most
positive: uses activator molecules that bind an activating binding site which encourages RNA polymerase association. signal molecule can bind the activator and cause to bind or unbind. In eukaryotes, activators bind Enhancer sequences. Eukaryotes use this regulation most.
describe the structure of operons
operons are polycistronic, they have multiple genes transcribed as a unit. consists of gene cluster, promoter, and additional regulatory sequences (eg activator binding site and repressor binding site)
describe the Lac operon
Lac operon encodes products that allow lactose import (lacY = galactosidase permease) and usage as fuel (lacZ = B-glactosidase) when glucose is not available. it only functions when lactose is present and glucose is absent.
negative control: lacI gene codes for repressor that blocks transcription of lacZ and lacY. when lactose is present, allolactose binds the repressor and releases DNA, allowing RNA polymerase to transcribe lacZ and lacY
positive control: CAP activator protein binds to CAP site on DNA and greatly increases lac operon gene expression. CAP can only bind if cAMP present, which occurs when glucose is absent as glucose inhibits adenylyl cyclase.
summary slide 15
two domains of proteins that bind DNA and examples
DNA/RNA binding domains: part of the protein must interact with nucleic acid (HTH, Zn fingers)
Protein-protein interaction domain: must interact with other proteins as well as nucleic acids (BHLH, Leu fingers)
how are specific DNA sequences recognized
major groove contacts and recognition of the pattern of H-bonding. the major groove is often the target because it has more room (can fit alpha helix better) and it has more potential H bonding patterns which creates more specificity. slide 17
common residues that contact DNA: Asn, Gln, Glu, Lys, Arg. basically the charged ones and N group ones
what is a Helix turn Helix (HTH) domain
a small DNA binding domain in prokaryotes and some eukaryotes. protrudes into major groove. often part of protein dimers. present in Lac repressor
what is a Zn finger domain
a small DNA (and RNA) binding domain in eukaryotes and few prokaryotes. consist of aa residues in a loop, help together by Cys or His interactions with a Zn2+. Zn stabilizes structure, does not contact DNA. there are often many Zn fingers in a single protein, not just one.
what are Leu zippers
protein binding domains present on DNA binding proteins, but does not bind DNA! consists of a Leu every 7th position on a helix that interact with each other and form a ‘zipper’ region
slide 20
wat is a basic helix loop helix (BHLH)
dimeric domains that bind proteins, present on DNA binding protein. dimeric species can occur between the same (homodimers) or similar (heterodimers) regulatory sequences. different dimers have different regulatory consequences important in neurodevelopment, myogenesis, hematopoiesis, etc
structure is larger than Leu zipper, has recognition helix, loop, and dimer-forming helices
describe transcriptional attenuation with trp operon as an example
slide 22-25
Trp repressor is signaled by high Trp levels to bind and block the trp operon, so no transcription occurs. With low and medium Trp levels, the attenuator is used to regulate.
medium high Trp levels: the ribosome translates sequence 1 and blocks sequence 2 before 3 is transcribed. as transcription continues, attenuation occurs by the structure formed by sequences 3 and 4. the attenuator (3/4 structure) causes strain and stops polymerase from transcribing.
low Trp levels: the low levels of TrptRNA causes the ribosome to pause and leaves sequence 2 unprotected. 2 interacts with 3 and no attenuator forms (3/4). transcription is not prevented!
describe regulation of prokaryotic protein levels by regulating translation
synthesis of ribosomal proteins is coordinated with rRNA synthesis. when an individual component of the ribosome becomes in excess of what is needed to make a ribosome, feedback inhibition occurs by translational repressor. the repressor is translated and then is blocks further translation of the same operon it came from
idk just feedback inhibition I think
describe regulation of prokaryotic protein levels by recombination events (flagellin proteins example)
relies on inverted repeats and transposition. The DNA for fljB and fljA is transcribed and translated to FljB flagellin protein and a FljA repressor (binds to FliC promoter, blocks FliC protein). But, the hin sequence (which has the FljB promotor) has inverted repeats and can be cut and reinserted into DNA facing the opposite direction. This means the promoter is now pointing the wrong direction so FljB and FljA are not made, and FliC is not repressed and makes the FliC flagellin protein
Basically: every so often the flagellin proteins swap from FljB to FliC and vice versa to avoid immune system recognition. the do this by flipping the promoter sequence around and changing transcription
slide 27
what is Cis regulation by prokaryotic riboswitches? TPP example
Cis = regulator is the same thing as what is being regulated.
the riboswitch is in the 5’UTR and has an aptamer and expression platform that fold together to affect genetic expression. The coding region of the gene is related in some fashion to the ligand that binds the aptamer (cis)
Aptamer = small length of nucleotides that fold into a specific 3D shape that is capable of binding a specific metabolite. aptamer is NOT a protein
how do riboswitches alter metabolite binding?
by negative feedback regulation. the aptamer is located upstream of the RBS and coding region. the coding region encodes enzymes needed for TPP (for example). Then TPP binds to aptamer and causes conformational change. this change decreases the gene product made by either stabilizing a transcription terminator or blockage of the RBS
slide 29
how and why design novel aptamers?
aptamers can be used to detect/bind compounds due to their specific binding capabilities. you can design novel aptamers with SELEX. first couple the desired ligand to the resin. generate tons of random RNA sequences and add to the column. most sequences will have no affinity and will flow through and discard. a few will have some affinity, collect these and amplify by PCR (allowing some variation) then re-apply the resulting RNA sequences to the column. Repeat the cycle until eventually you have selective RNA with an affinity for the ligand!
aptamers as therapeutic agents?
they can function similar to antibodies as drugs with specific recognition of targets. they are smaller and more economic than antibodies
aptamers as detectors?
aptamers detect the binding of a specific ligand. this can be monitored by electrical sensors or fluorescence based sensors. electrical works by placing an electron donor on the aptamer which will be brought close to a sensor when the aptamer binds its ligand and changes conformation. the electron is donated to sensor and so measures the amount of ligand.
fluorescence is similar, works by including a dye and a quencher molecule on the aptamer which are moved apart when aptamer is ligand bound (or vice versa). Can also use FRET for same idea
slide 33
what is trans regulation by prokaryotic RNA/proteins? rpoS example
trans = a different product regulates than the molecule that is being regulated. rpoS mRNA has the RBS blocked and translation cannot begin. Under stress conditions, DsrA RNA is induced (encoded by a totally separate gene) and binds along with Hfq to open up the RBS. Now translation can occur of sigma factor which helps express genes to combat stress
slide 34
how are unnatural amino acids incorporated in prokaryotic ribosomes?
mutate a tRNA synthetase to accept a tRNA containing an anticodon for UAG amber stop codon and an unnatural amino acid. then mutate the codon in the ORF that you want to replace with the unnatural amino acid with the UAG codon. recall that the ribosome does not check the tRNA aa for correctness, it will just incorporate it.
canonical = normal tRNA and normal aa
orthogonal = mutated tRNA and aa that still works as a unit
differences between eukaryotic vs prokaryotic gene expression regulation
eukaryotic shows more weak interactions between RNA polymerase and promoter, so require factors to be turned on, using a lot more positive regulation.
eukaryotes have restricted access due to chromatin
eukaryotes have larger more complex multimeric regulatory proteins and more regulatory sequences, often 100s of bp away from promoter. a single gene may be regulated by several proteins
transcription is separated from translation by space and time
describe eukaryotic gene/RNA structure
genes have regulatory sequences, promoters, and non-coding sequences in 5’, 3’, and within the gene (introns). Exons are coding for protein. introns are noncoding but have regulatory roles (eg miRNA precursors)
RNA has 5’ UTR which is recognition site, AUG start and stop codons around the coding exons, and 3’ UTR that has polyA tail (also recognition)
how is eukaryotic transcription regulated by RNA polymerase finding/binding promoter? things that affect polymerase binding
- Regulatory DNA sequences: -30 TATA box and lots of other wayyy upstream sequences
- how the genes are compacted: chromatin remodeling allows polymerase to bind or not bind
- regulatory proteins and RNAs: certain transcription factors, coactivators, long non coding RNAs, and miRNAs affect RNA polymerase binding
describe chromatin remodeling as a regulator of transcription
10% of chromatin is more condensed heterochromatin, transcriptionally inactive. less condensed is euchromatin. transcriptionally active regions are distinguished by:
1. the positioning of the nucleosomes
2. the presence of histone variants
3. the covalent modifications of nucleosome histones
changing these 3 things remodels chromatin structure. Remodeling employs enzymes that unwrap, translocate, remove, or exchange, nucleosomes
how is chromatin remodeled by histone replacement?
Histone chaperones, which are specific for their particular histones, can exchange histones for variants which alters transcription. The composition of histones is related to the level of transcription occurring
how is chromatin remodeled by nucleosome positioning (histone modifications)?
methylation of DNA and histones causes tighter packing which prevents access by transcription factors, genes are not expressed. Methylation = silencing
acetylation of histones causes loose packing and allows access of transcription factors, genes are expressed. Acetylation = inducing
how is chromatin remodeled by histone modifications?
histones can be modified in tons of places in many different ways which will affect transcription. this is done by enzymes:
writers are the enzymes that add modifications like acetylations, methylations, phosphorylations, etc on histone tails
erasers are enzymes that remove modifications
readers are enzymes/proteins that bind the specific code
describe HATs and HDACs
histone acetyltransferase (HAT) is a writer that acetylates Lys on the histone tail histone deacetyltransferase (HDAC) is an eraser that removes the acetyl group on the Lys acetylation reduces the positive charge on Lys residues and destabilizes interactions between tails and structural proteins. acetyl group makes more transcriptionally active
describe methyltransferases
methyltransferases (MT) can modify Lys (KMT) and Arg (PRMT) residues on histone tails. both are writers which add methyl groups. They can add the groups in a few ways to make mono, di, or tri methyl lysine/arginine.
describe epigenetic in context of histones
epigenetics: changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself. This modifications include histone patterns like methylations or acetylations
epigenetic programs can be inherited for several generations. changes can be induced by chemicals
what is LncRNA? functions?
long noncoding RNA: greater than 200 nucleodiyes in length with little to no protein coding potential.
they resemble mRNAs as they are transcribed by RNA pol II, 5’ capped, 3’ polyadenylated, and splice.
Functions:
guides by binding proteins and directing them to a DNA/RNA sequence that is complementary
dynamic scaffolds that work like adaptors and bind 2 complexes, bringing them close together and encouraging interaction
molecular decoys that bind transcription factors or miRNAs and prevent them from interacting with their real target
types of molecules usually required for RNA pol II binding at promotor
basal transcription factors: needed at ALL promoter sites
DNA binding transactivators (transcription factors): bind enhancers and facilitate transcription
coactivators: do NOT bind DNA but interact with transcription factors to facilitate transcription (adapters)
architectural regulators: bend DNA (eg high mobility group proteins)
corepressors: prevent/block transcription
enhancers: regulatory sequences that area often many bp away from the promoter. means that a single gene may be regulated by several proteins
describe eukaryotic DNA transactivators
also called transcription factors
each will have 2 domains, a protein interaction domain and a DNA binding domain. They bind to enhancers, which may be very far away from promoters, and interact with promoters via looping/bending of DNA promoted by specific architectural proteins such as HMG
they differ between cells, can be inactive or active, often PTMed, usually are dimeric to be active, and can be transported from cytoplasm to nucleus to effect activity
describe eukaryotic coactivators
they DO NOT directly interact with DNA, though they often interact with the CTD of RNA Pol II. They may function to stabilize the ‘active’ dimeric form a transcription factor or act as an adaptor or scaffolding for other proteins
define mediator
a large complex of 20-30 proteins that acts as an intermediate between the transcription factors and RNA polymerase. Basically its all of the extra junk that is needed to promote transcription and transmit all of the signals from transcription factors, enhancers, etc
what are common ways that transcription factors are activated?
PTMs and binding of small molecules (hormones!)
slide 16, I don’t really understand. watch again?
how can the same protein act as a transactivator and a repressor?
it depends on the presence of a hormone in some cases. proteins will bind the hormone signal and have some kind of conformational change which allows them to interact with specific consensus sequences for called Hormone Responsive Elements (HREs) in the DNA. The binding then regulates transcription of the adjacent genes
slide 17
two types of steroid hormone signaling
type I: the receptors are located in the cytoplasm. hormone binds receptor, causes conformational change allowing the protein to enter nucleus and bind HRE, interacts with transcription complex
type II: receptors are located in the nucleus. the hormone enters the cell and then the nucleus where it interacts with proteins bound to the HRE and affects transcription
describe the structure of a typical receptor for steroid hormone signaling
Has a transcription activation sequence (protein interaction domain), DNA binding domain, and hormone binding domain. may have Zn fingers to bind DNA. often susceptible to regulation by phosphorylation or other PTMs
summary of regulation of eukaryotic translation
- initiation factors: subject to phosphorylation (=less active) and can cause rapid activation of translation by phosphatase mediated mech
- translation repressors: proteins that bind to the mRNA and prevent translation
- binding proteins: disrupt the interaction of eukaryotic translation factors. regulation by phosphorylation
- miRNA: degrades the translation template or directly blocks translation
phosphorylations interfere with translation because they interfere with the binding of the tail and cap with the ribosomes. remember that they are all tied up together
