Flashcards in 8.18.16 Lecture Deck (57):
Transcription cannot occur and regulate gene expression if...
...we cannot also regulate the downstream products of transcription.
What are the post-transcriptional steps that control gene expression that occur in the nucleus?
2. RNA processing control, alternative splicing, capping, cleavage/polyadenylation
3. RNA editing
What are the post-transcriptional steps that control gene expression that occur in the cytoplasm?
1. Translation control, localization control, translation initiation
2. mRNA stability
3. Protein control, turnover, modification, and inhibition
Alternative splicing can lead to over ___ proteins.
What are the four common types of alternative splicing?
1. Exon skipping
2. Alternative 3' splice site selection
3. Alternative 5' splice site selection
4. Intron retention
What are the three uncommon types of alternative splicing?
1. Mutually exclusive exons
2. Alternative promoters
3. Alternative polyadenylation
Describe the way alternative splicing affects the fibronectin gene.
Fibroblast fibronectin and hepatocyte fibronectin are encoded by the same gene. Fibroblasts synthesize mRNA that contains the exons EIIIA and EIIIB, which encode protein regions responsible for binding the cell membrane. Hepatocytes splice out these exons.
What is the different between fibroblast fibronectin and hepatocyte fibronectin?
Fibroblast fibronectin is insoluble and is a part of the extracellular matrix that binds to the cell membrane; it is involved in cell adhesion. Hepatocyte fibronectin is soluble and circulates in serum; it is important in clot formation.
The Drosophila DSCAM gene can produce ___ different splicing patterns.
Variants of the DSCAM gene contribute to what functions in Drosophila?
1. Plasticity of the immune system and formation of complex neural circuits
2. Mediate phagocytosis of bacterial pathogens
How do regulatory proteins aid in the selection of splice sites?
Regulatory proteins either promote or block the use of specific splice sites by binding to regulatory sequences in pre-mRNA; called splice site enhancers and silencers, respectively.
Describe negative control of splice site selection.
A strong splice signal will likely be used no matter what, unless it is inhibited. A repressor interacts with a silencer sequence and discourages splicing. This is NOT a physical blockade.
Describe positive control of splice site selection.
A weak splice signal will likely be ignored unless it is enhanced. An activator interacts with an enhancer sequence and encourages splicing.
SR proteins recruit ___ components to 5' and 3' splice sites. How does this function in genes that cannot do alternate splicing?
Spliceosome; these proteins ensure correct splicing in instances without possible alternative splicing
SR proteins are rich in what two amino acids?
Ser and Arg
What is the difference between constitutive splicing and alternative splicing?
In alternative splicing, the levels of the activator and repressor proteins vary between tissues.
The choice of ___ and ___ sites in a pre-mRNA can affect the protein product of a gene.
Cleavage; polyA sites
Describe regulation of 3' end processing of immunoglobulin mRNAs in B cells.
In a resting B cell, a long RNA transcript is produced and splicing of the intron occurs; the cell uses the strong splice site. The membrane-bound antibodies are produced. In an activated B cell, the weak site is used, the transcript is cleaved, and polyadenylated upstream of the splice site. This leads to the hydrophilic portion of the secreted antibody.
___ CstF leads to a resting B cell. ___ CstF leads to an activated B cell. CstF interacts with the ___ site.
Decreasing; increasing; poly-A.
In eukaryotic cells, the ___ of translation initiation is more common than the regulation of individual mRNAs.
What leads to inhibition of protein synthesis in regulation of translation?
Phosphorylation of eIF-2
Describe the eIF2 cycle.
1. GDP-bound eIF2 is inactive.
2. Inactive eIF2 binds eIF2B
3. GDP is removed from eIF2*eIF2B and GTP is added.
4. GTP-bound eIF2 is active.
When inactive GDP-bound eIF2 is phosphorylated by a kinase, it binds and sequesters eIF2B.
What is the cap-binding protein?
Describe the regulation of eIF4E.
eIF4E is activated by growth factors resulting in phosphorylation of eIF4E-BP (binding protein), an eIF4E inhibitor. This phosphorylation causes eIF4E-BP to leave; eIF4E is then able to bind eIF-4G, which is required to recruit the small ribosomal subunit. Dephosphorylation of eIF4E-BP (triggered by serum deprivation, viral infection, heat shock, and M phase) occurs. eIF4E-BP binds to eIF4E, preventing its binding with eIF4G and inhibiting synthesis.
Some mRNAs contain ___ with features that inhibit translation.
5' untranslated regions (5'-UTRs)
What is an inefficient mRNA?
Inefficient mRNA contains long 5'-UTR with extensive secondary structure of AUG codons upstream of the translation start site.
What happens to inefficient mRNA when eIF4E levels are low? What happens to efficient mRNA when eIF4E levels are low?
It is poorly translated; it is translated well.
Inefficient mRNA typically encodes proteins involved in cell growth and cell cycle regulation. Why?
This helps protect against cancer, since these genes can be considered proto-oncogenes.
mRNA containing an IRES can be...
...translated independent of a N7-methylguanine cap. IRES allows the 40S subunit to bypass the cap when forming a functional translation initiation complex with the corresponding mRNA. Thus, inhibition of eIF4 by eIF4-BP does not stop translation of mRNAs containing IRES. In fact, inhibition of eIF4 by eIF4-BP likely activates translation of mRNAs containing IRES.
Give an example of the use of IRES.
Activation of apoptosis; initiated by caspases, which target eIF4G. This turns off synthesis of many cellular proteins while activating synthesis of proteins needed for cell death. These mRNA likely contain IRES.
mRNA stability varies from many hours to less than 30 minutes depending on the mRNA. The amount of a given mRNA in a cell is a ___ that is related to its rate of ___ and rate of ___.
Steady state; synthesis; degradation
The time required for mRNA to reach its new level depends only on ___.
The rate of degradation
If an mRNA or protein must be regulated rapidly, it will tend to have a high ___ rate. This means it has a ___ half-life and is ___.
Degradation; short; unstable
___ of mRNA triggers degradation.
Describe the process of deadenylation.
Deadenylation begins slowly and is triggered by loss of poly-A binding proteins. When the poly-A tail is shortened to
There is competition between mRNA translation and mRNA decay. Both deadenylase and translation initiation machinery associate with ___ and ___.
5' cap; poly-A tail
What are miRNAs?
MicroRNAs; non-protein coding RNA products of miRNA genes, usually found in introns or non-protein coding regions between genes, are capped and poly-adenylated, have distinctive double strand structure, mainly function to reduce or silence expression of protein coding genes, synthesized by RNA polymerase II
Describe the mechanism of miRNA gene expression regulation.
1. Precursor miRNA forms a double-stranded structure.
2. miRNA cropped in nucleus, exported to cytosol
3. Cleaved further by Dicer to the miRNA proper
4. One strand is degraded; the guide strand remains
5. Guide strand moves RISC to specific mRNAs via base pairing.
6. If match is extensive, rapid degradation of mRNA occurs. If the match is ~7 nucleotides (seed), translation is inhibited, mRNA is destabilized, and mRNA is transferred to P-bodies. This is degraded eventually.
Why can 1 miRNA bind to many transcripts?
Cancer uses ___ to try to survive.
What is nonsense-mediated mRNA decay?
Special pathway for eliminating mutant and aberrant mRNAs that contain a premature stop codon.
Describe the process of nonsense-mediated mRNA decay.
mRNA are marked by exon junction complexes (EJCs) when spliced successfully. As the mRNA passes through the tight channel of the ribosome, the EJCs are stripped off and successful mRNA are released. If an in-frame stop codon is encountered before the final EJC, the mRNA undergoes nonsense-mediated decay, triggered by UpF proteins that bind to each EJC.
___ tags proteins for degradation by the proteasome.
How does ubiquitin bind to its target?
C-terminal Gly covalently attaches to a Lys in protein destined for degradation
What degrades the protein?
Describe the ubiquitination pathway.
1. E1, the ubiquitin activating enzyme, transfers ubiquitin to E2, the ubiquitin conjugating enzyme.
2. E2 binds to E3, the ubiquitin protein ligase, which is bound to the target protein.
3. Ubiquitin(s) placed on the target protein.
4. Protein transferred to proteasome; ubiquitin recycled
What are the ubiquitin enzymes and how many of them are there in a cell?
E1 - ubiquitin activating enzyme, 1
E2 - ubiquitin conjugating enzyme, 20
E3 - ubiquitin protein ligase, over 500
Describe the structure and function of proteasome.
The proteasome is composed of a central 20S cylinder (proteases found here) supplemented by 2 19S caps. The complex cap selectively binds ubiquitinated proteins; uses ATP hydrolysis to unfold chains and feed them into the inner chamber of the 20S cylinder for digestion into short peptides.
What are some of the proteins regulated by ubiquitin-dependent proteolysis?
-Cell cycle regulatory proteins
-Apoptotic regulatory proteins
-Components of signal transduction pathways
-Components of mitotic machinery
-Abnormal or damaged cellular proteins
___ have destruction box sequences targeted by ubiquitin enzymes.
Describe the relationship between NF-kappa-B and I-kappa-B.
NF-kappa-B is a transcription factor that activates cell growth and division. Normally, I-kappa-B binds NF-kappa-B and prevents it from signaling. When I-kappa-B is phosphorylated, it releases NF-kappa-B. I-kappa-B is then polyubiquitinated and degraded. This can lead to cancer.
Blocking the ___ may be a way to treat cancer.
Mutations in ___ or their protein substrates are associated with disease.
E3 ubiquitin ligase
In Parkinson's disease, ___ is mutated. ___ is a substrate for this and can produce insoluble protein aggregates in the brain.
Parkin, an E3 ubiquitin protein ligase; Alpha-synuclein
In hereditary tumors of retina, brain, kidney, and other tissues, ___ is mutated. This is a component of E3 ligase complex that targets HIF, a TF promoting angiogenesis.
In hereditary breast and ovarian cancer, ___ and ___ are mutated. These are components of E3 ubiquitin ligase that activate proteins involved in DNA repair.