Mol Lecture #16 Flashcards
Eukaryotic Gene Regulation
- Lifespan can also be regulated
- Key aspects: Multiple Levels
- Transcriptional regulation: genes can be co-regulated but do not exist in operons. (No operons in eukaryotes)
- More complex regulation in transcription
- Transcriptional: basal transcriptional control (basic things needed to transcript) v.s. Specific transcriptional control (specific factors needed)
Eukaryotic Organization
- Enhancers (promoter proximal enhancers- involved in specific regulation)
- Promoter is basal transcription
Basal transcription
- Promoter (Tata box): recruit general transcription factors
–> General transcription factors are going to further recruit RNA polymerase II
–> Gen transcription factor unwinds promoter DNA and transcription begins.
Specific transcription
- Second level of regulation that allows basal transcription to occur in 1) tissue/ cell-specific manner OR 2) stimulus-responsive manner
- Requires multiple regulatory sequences in the DNA bound by sequence-specific DNA binding proteins
→ specific transcription factors
2 major mechanisms to increase transcription
1) modulate DNA packing on the histones
2) increase or decrease recruitment of RNA polymerase II
Enhancers and Transcription Factors
- Unique sequences of enhancers
- Sequences: specific sequences of DNA that allow for binding of Txn (transcription) factors
- Activators bind to enhancers
- Regulatory sequences are referred to as cis-elements- bound by specific transcription factors (activators- can have positive or negative effects on transcription)
Long-Range Interactions
- Working with coactivator complex to build a bridge to bridge enhancers- creates a DNA loop.
- Allows activators to interact with each other.
Histones and Regulation of Gene Expression:
Chromatin remodelling
move or remove histones to alter local density (low density)
- Using histone modifications can make DNA more accessible via relaxation
- Using acetyltransferase, we add acetyl groups to relax the tails: histone modifications increase accessibility by relaxing tails.
Regulation at the level of mRNA
- 2nd level of control that can allow more rapid responses
*mRNa processing - mRNA stability
mRNA processing
regulate splicing, alternative splicing
mRNA
changing poly A tail addition or 5’ cap
–> Have mRNA regulation in 5’ and 3’ untranslated regions (UTR)
RNAi: RNA interference
- noncoding single-stranded RNAs bind to target mRNAs, affecting their stability and translation.
- miRNA
MicroRNA (miRNA): Gene regulation
- endogenous RNAs transcribed from the genome
- pre-miRNA has a stem-loop structure (hairpin) (made up of self-base pairing)
–> dicer (protein) will essentially cutoff the hairpin (turning into two molecules)
–> RISC is looking for the appropriate mRNA that has close base-pairing with the miRNA (base pairing of miRNA w/ target mRNA influences stability + translation of mRNA)
–> Short interfering RNA (siRNA)
RISC
*miRNA-induced silencing complex
siRNA
- produced from double-stranded RNA that is not encoded by the genome
- Helps to silence gene of interest
Regulation of translation
- RNA binding protein
- mRNA sequence + structure to interact w/ proteins
- Stem-loop structures in 5’ + 3’ (UTR) directly control ribosome scanning and translation initiation (or via binding to RNA interacting proteins)
Regulation of translation example
- Ex. Ferritin: Iron binding protein used to store iron (free iron is toxic)
→ In the absence of iron, ferritin is not going to be translated
Post-translation regulation
- Covalent modifications (phosphorylation, acetylation) are used to change protein stability, activity, function, and localization)
Localization
- proteins need to get to location of activity
→ in eukaryotic cells, getting a protein to the right place is challenging because we have different places (nucleus, cytoplasm, endomembrane system). Therefore, proteins will have a specific amino acid motif called localization proteins telling them where they need to go.
Processing of proteins
- cleavage of proteins (cutting it up) to change function
Degradation
- protein levels and therefore activity can be altered by degrading proteins. (get rid of damaged proteins)
→ protein levels can be regulated by controlling the half-life or stability of a protein.
→ Tag proteins with ubiquitin which then directs them to be degraded by the proteasome.
Discovery of the Nuclear localization signal (ex., Of a type of nuclear localization signal- motif)
- SV40 large T antigen: when expressed in cells it would always end up in the nucleus
- N terminus and C terminus
- The nuclear localization sequence was in the 127-133 set of amino acid sequence.
- A specific set of amino acids tells the protein where it needs to go.
Point: Forcing GFP to go to a different site by adding new sequence.
Ubiquitin and the Proteasome
- Covalently add Ubiquitin
- Large protein complex with proteolytic activity (helping to degrade proteins)
