Mol Lecture #16

Question 1

Eukaryotic Gene Regulation

Answer 1

• 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)

Question 2

Eukaryotic Organization

Answer 2

• Enhancers (promoter proximal enhancers- involved in specific regulation)
• Promoter is basal transcription

Question 3

Basal transcription

Answer 3

• 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.

Question 4

Specific transcription

Answer 4

• 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

Question 5

2 major mechanisms to increase transcription

Answer 5

1) modulate DNA packing on the histones
2) increase or decrease recruitment of RNA polymerase II

Question 6

Enhancers and Transcription Factors

Answer 6

• 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)

Question 7

Long-Range Interactions

Answer 7

• Working with coactivator complex to build a bridge to bridge enhancers- creates a DNA loop.
• Allows activators to interact with each other.

Question 8

Histones and Regulation of Gene Expression:
Chromatin remodelling

Answer 8

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.

Question 9

Regulation at the level of mRNA

Answer 9

• 2nd level of control that can allow more rapid responses
  *mRNa processing
• mRNA stability

Question 10

mRNA processing

Answer 10

regulate splicing, alternative splicing

Question 11

mRNA

Answer 11

changing poly A tail addition or 5’ cap
–> Have mRNA regulation in 5’ and 3’ untranslated regions (UTR)

Question 12

RNAi: RNA interference

Answer 12

• noncoding single-stranded RNAs bind to target mRNAs, affecting their stability and translation.
• miRNA

Question 13

MicroRNA (miRNA): Gene regulation

Answer 13

• 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)

Question 14

RISC

Answer 14

*miRNA-induced silencing complex

Question 15

siRNA

Answer 15

• produced from double-stranded RNA that is not encoded by the genome
• Helps to silence gene of interest

Question 16

Regulation of translation

Answer 16

• 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)

Question 17

Regulation of translation example

Answer 17

• 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

Question 18

Post-translation regulation

Answer 18

• Covalent modifications (phosphorylation, acetylation) are used to change protein stability, activity, function, and localization)

Question 19

Localization

Answer 19

• 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.

Question 20

Processing of proteins

Answer 20

• cleavage of proteins (cutting it up) to change function

Question 21

Degradation

Answer 21

• 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.

Question 22

Discovery of the Nuclear localization signal (ex., Of a type of nuclear localization signal- motif)

Answer 22

• 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.

Question 23

Ubiquitin and the Proteasome

Answer 23

• Covalently add Ubiquitin
• Large protein complex with proteolytic activity (helping to degrade proteins)