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57 Cards in this Set

  • Front
  • Back
Tissue specific gene expression
housekeeping genes, specialized genes, and finely tuned genes
housekeeping genes
always on (e.g. ribosomal proteins, polymerases, DNA repair enzymes)
specialized genes
either on or off (hemoglobin in red blood cells)
finely tuned genes
most genes; can change in response to external signals (e.g. starvation causes the synthesis of specialized enzymes in the liver which convert amino acids to glucose)
levels of gene expression
-transcriptional control
-Post-transcriptional control – includes RNA processing (splicing, capping, adenylation, RNA transport and localization, RNA degradation)
-translational control
-post-translational control
Transcriptional control
complex due to large genome size and multiple types of cells, includes:
1. RNA polymerase II cannot begin transcription on its own, requires general transcription factors, provides multiple steps where transcription rate can be controlled, gene regulatory proteins regulate these steps and therefore the rate
2. dense packaging into chromatin
3. regulatory sequences can affect the promoter
general transcription factors
assembled at the promoter before transcription can start, general because they assemble at all promoters transcribed by RNA polymerase II
gene regulatory proteins
act only at a specific gene, determining if it is on or off
genetic switches
transcriptional control, are DNA binding proteins that bind to specific DNA nucleotide sequences, used to turn genes on or off
components of a genetic switch
1. specific DNA sequences, generally 8 - 15bp long
2. the proteins that bind to them
negative control/regulation
occur when proteins bind to DNA and turn genes off, the gene regulatory proteins that act in this manner are called transcriptional repressors or gene repressor proteins
positive control/regulation
occur when proteins bind to DNA and turn genes on, the gene regulatory proteins that act in this manner are called transcriptional activators or gene activator proteins
tryptophan operon
example of a simple switch that is regulated by the food in the environment, when tryptophan is present in the environment, the cell no longer needs to make it so the genes for its synthesis are turned off, therefore negative control, need two tryptophan bound to the operator to inhibit
genes adjacent to one another on the chromosome and are transcribed from a single promoter as one long mRNA molecule
a region of regulatory DNA (consensus sequence) that is recognized by a repressor protein, if the repressor is bound to the operator it blocks access to the promoter by the RNA polymerase
lac operon
under both negative and positive control, lac repressor protein and CAP (catabolite activator protein), operon is expressed only if lactose is present (which removes the lac repressor) and glucose is absent, remain in off state unless this is met
CAP (catabolite activator protein)
activates genes in E. coli to use alternative energy sources (such as lactose) when glucose is not available
lac repressor
ensures that the lac operon is off if lactose is not present
DNA sites where gene activators would bind to enhance transcription
how do gene regulatory proteins affect the rate of transcription when they can (a) act 1000s of nucleotides away from the promoter or (b) be upstream or downstream?
the DNA between the enhancer and the promoter loops out to allow the activator proteins bound to the enhancer to come into contact with proteins (RNA polII and/or transcription factors) bound to the promoter, DNA acts as a tether
what does a eukaryotic gene control region consist of?
the promoter and the regulatory DNA sequence, assembled as a complex that can act as an activator or repressor
coactivators and corepressors
do not bind to DNA themselves, but assemble of DNA bound gene regulatory proteins to activate or repress respectively
main function of activators
attract, position and modify the general transcription factors and RNA polymerase II to begin transcription, can also change the chromatin structure of the regulatory sequence and promoter
how is chromatin structure altered?
1. covalent histone modification
2. nucleosome remodeling
gene activating proteins and chromatin structure
use both mechanisms by recruiting histone acetyl transferases (HATs) and ATP dependent chromatin remodeling complexes
covalent histone modification
one of the key regulatory mechanisms in transcription regulation, acetylate, methylate, phosphorylate and ubiquitinate, these allow greater accessibility to the underlying DNA which facilitates the assembly of the general transcription factors and RNA polymerase II on the promoter, also allows for binding of gene regulatory proteins
gene repressor proteins and chromatin structure
inhibit binding of general transcription factors and RNA polymerase through histone de-acetyl transferases (HDATs)
what is combinatorial gene regulation?
the ability of multiple gene regulatory proteins to assemble in different ways to regulate the transcription of different genes, leads to the development of different cell types
importance of combinatorial gene regulation
emphasizes the complexity of cellular and tissue development and differentiation to create specialized cell types
activation and repression of genes and tumorigenesis
important in trying to find new ways to specifically treat diseases
Post-transcriptional control
1. transcriptional attenuation
2. alternative splicing
3. RNA cleavage
4. RNA stability and degradation
5. RNA transport
6. mRNA localization
transcriptional attenuation
used by genes that are continuously transcribed, early termination of transcription, based on mRNA structures reaction with RNA polymerase II
alternative splicing
occurs because of intron sequence ambiguity, spliceosome is unable to differentiate between the different splice sites, different choices made by chance, types of alternative splicing include: cryptic splice site, exon skipping, can be positively or negatively regulated as well
cryptic splice site
A site whose sequence resembles an authentic splice site and which might be selected instead of the authentic site during aberrant splicing
exon skipping
aberrant splicing when one or more exons are omitted from the spliced RNA
why is alternative splicing important
increases the diversity of proteins produced from a single transcript, ex: tropomyosin in different muscle cells
conserved motifs for splicing (exon-intron junction)
GU at the 5’ end and AG at the 3’ end, known as the GUAG rule
RNA cleavage
cleavage at different positions on RNA transcript alter the localization of the resulting protein, membrane bound vs. secreted antibodies
RNA interference (RNAi)
post-transcriptional gene silencing when double stranded RNA is introduced into a cell causing sequence specific degradation of homologous mRNA, used to treat disease
mechanism of RNAi
1. dsRNAs inserted into the cell and initiate RNAi
2. first processed by Dicer (ATP dependent RNAse III enzyme)
3. dicer process dsRNAs into short interfering RNA (siRNA, 21-23 nt long)
4. The siRNAs are incorporated into the RNA-inducing silencing complex (termed RISC). The sense strand of the siRNA duplex is cleaved leading to activation of the RISC
5. The remaining antisense strand of the siRNA guides RISC to its homologous mRNA, resulting in the cleavage of the target mRNA
AKA short interfering or silence RNA, can be synthesized artificially and then introduced into the cell to inhibit transcription, can also act in altering chromatin structure
micro RNAs (miRNA)
negatively regulate gene expression, target mRNA for cleavage or translational repression, a very abundant gene regulatory molecule, influence output of protein-coding genes, single stranded that are transcribed but not translated, has a reverse complement, base pairs with its reverse complement to form a hairpin loop (pri-miRNA
found in the nucleus, cleaves pri-miRNA to form pre-miRNA
carrier protein that carries pre-miRNA out of the nucleus into the cytosol
Dicer and miRNA
cleave the pre-miRNA to form 20-25 nt molecule
how does miRNA work?
thought to block the protein translation machinery, may also target methylation of genomic DNA, works in conjunction with micro ribonucleoprotein (miRNP) which is similar to RISC
RNA transport
involvement of the 3’ –polyA tail and 5’-cap 7mG, only a correctly processed RNA transcript will be transported
mRNA localization
Mechanism unknown, but thought there is involvement of 3’ UTR. Results in association of the mRNA transcript with ribosome in the cytosol or the endoplasmic reticulum
Translational control of gene expression
1. mRNA stability and degradation
2. translation initiation
3. translation efficiency
mRNA stability and degradation
if mRNA unstable then don’t make anymore protein from it
what determines if an mRNA is stable?
1. length of poly-A tail
2. the sequence in the 5’ and 3’ untranslated region (UTR)
responsible for deadenylation dependent mRNA decay, it shortens the poly-A tail
translation initiation
1. kozak sequence
2. eukaryotic initiation factors
kozak sequence
A/GxxAUGG, optimal for translation initiation, usually AUG at 5’ cap, can be blocked by repressor proteins that hide the kozak sequence
eukaryotic initiation factors (eIFs)
important for translation initiation, some bind to the ribosomal subunit while others associate to the Met-tRNA, some recognize and bind to the 5’ cap and poly-A tail, bring the mRNA to the ribosome
translation efficiency
elongation factors EF-1 and EF-2 and their association with GTP and hydrolysis to GDP controls translation accuracy and efficiency
Post-translational control of gene expression
1. control of protein folding and association with other protein subunits
2. protein processing (cleavage to form an active form)
3. phosphorylation and dephosphorylation that can activate or inactivate a protein
4. compartmentalization of proteins into particular regions of the cell
5. protein degradation (ubiquitination), balanced with synthesis