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MCB 2050 > Lecture 5 > Flashcards

Lecture 5 Flashcards

1
Q

What do protein binding transcription control regions do?

A

Regulate expression of euk genes, they are located at multiple sites at various distances from the txn start site

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2
Q

What are the two types of protein binding txn control regions?

A

Promoter proximal elements and enhancers

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3
Q

What are promoter proximal elements and enhancers?

A

Gee and cell-type-specific regulatory elements on DNA

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4
Q

What are transcription activators and repressors?

A

Modular proteins containing a DNA binding domain and one or more activation or repression domains

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5
Q

How do we find gene regulatory sequences?

A

Link scanning reporter gene technique for identifying gene regulatory elements on DNA

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6
Q

What is linker scanning mutagenesis?

A

A way to find gene regulatory sequences.

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7
Q

What are the steps to linker scanning mutagenesis?

A
  1. Buy a plasmid that contains a reporter gene, usually a gene that encodes an enzyme
  2. The ORF of the reporter gene (tk) on the plasmid has no promoter
  3. Upstream of the tk ORF you clone a large piece of DNA that you know is controlling your gene (not tk) of interest
  4. Replace small pieces of the cloned regulatory DNA by a “linker”, produce multiple plasmids with linker subs
  5. Transform each individual plasmid in a cell culture and measure the activity of the reporter
  6. Loss of reporter activity means that you have hit a regulatory sequence
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8
Q

What is a reporter gene?

A

DNA sequence that encodes for protein sequence that we recognize

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9
Q

What do reporter plasmids contain?

A
  1. An ORF for an enzyme
  2. No promoter proximal regulatory sequences
  3. Multiple cloning site (MCS) for the cloning a regulatory sequence
  4. Translation initiation elements upstream of the reporter gene
  5. Poly A+ signal downstream of the reporter gene
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10
Q

What are some control elements that regulate gene expression in euk? TATA vs CpG islands

A

TATA promoter
Promoter elements: position RNA pol II to initiate txn at the start site, influence the rate of txn
Enhancers: can be upstream or downstream or in introns
Promoter-proximal elements: found upstream and downstream of txn start sites
CpG island promoter
Txn initiates at several sites in both the sense and antisense directions from the ends of the CpG-rich region
Sense direction transcripts are elongated and processed into mRNA by RNA splicing
Genes express mRNAs with alternative 5’ exons determined by the txn start site
Promoters contain promoter-proximal control elements

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11
Q

What are some characteristics of enhancers?

A

Can be far from the promoter, bend chromatin/DNA, orientation doesn’t matter, can be downstream or upstream, typically on the same chromosome

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12
Q

Enhancers are ___ regulatory elements

A

Distal

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13
Q

What are the main differences between enhancers and promoters?

A

Promoters function within a short distance while enhancers function over a long distance

Promoters are immediately upstream from the initiation site (RNA Pol II) while enhancers can be upstream, downstream from the start or within introns

Promoters are position-dependent which enhancers are position-independent

Promoters are orientation-dependent while enhancers are orientation-independent

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14
Q

What inactivates transcription?

A

Chromatin condensation blocks RNA pol and GTFs from interacting with promoters

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15
Q

What are the two things that repressor proteins can do?

A

Bind to txn control elements to inhibit txn initiation
Interact with multi protein co-repressor complexes to condense chromatin

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16
Q

What is are the functions of a pioneer transcription factor?

A

Binds to a specific regulatory sequence within the condensed chromatin
Interacts with chromatin remodeling enzymes and histone acetylases that decondense the chromatin making it accessible to rna pol II and gtfs

17
Q

What is the function of activator proteins?

A

bind to specific transcription control elements in both promoter-proximal sites and distant enhancers
interact with one another and with the multisubunit mediator complex to assemble gtf’s and rna pol II on promoters

18
Q

What is a domain?

A

part of a polypeptide with a specified function

19
Q

What are four domains?

A

Transactivation domain, dimerization domain, dna binding domain, hormone binding domain

20
Q

How are domains identified?

A

Deletion analysis of peptides

21
Q

What are the steps to deleting mutants of the GAL4 gene in yeast?

A
  1. reporter gene containing lacZ reporter gene and TATA box ligated to UAS GAL (regulatory element) that contains Gal4 binding sites
  2. reporter gene construct and dnas encoding wild type or mutant gal4 introduced simultaneously into mutant gal4 yeast cells
  3. activity of beta galactosidase expressed from lacZ assayed (high activity if introduced gal4 DNA encodes a functional protein
  4. DNA binding site: eliminates binding to UASgal and beta galactosidase activity
  5. Activation domain: only the beta galactosidance activity eliminated
22
Q

What is a motif?

A

conserved sequence in a protein found in a domain that is likely to produce a distinct 3D structure and a distinct activity in vivo

23
Q

How many activation and dna binding domains do tf’s usually have?

A

TF’s may contain more than one activation domain but rarely contain more than one DNA binding domain

24
Q

What domain does the Zn finger bind to?

A

DNA binding domain

25
Q

What domain does the Leu zipper motif bind to?

A

Dimerization domain

26
Q

What domain does the hormone binding motif bind to?

A

Hormone binding domain

27
Q

C2H2 Zn finger characteristics?

A
  • contains two conserved cysteine and two conserved histidine residues, whos side chains bind to one zn ion
28
Q

C4 Zn finger characteristics?

A
  • four conserved cysteins bind zn
  • bind DNA as homodimer - one alpha helix in each monomer interacts with the dna
29
Q

Leucine zipper protein motif characteristics?

A
  • basic residues in th extended alpha helical regions on the monomers interact with the DNA backbone at adjacent sites in the major groove
  • coiled-coil dimerization domain is stabilized by hydrophobic interactions between the monomers
  • can form heterodimers
30
Q

What are the characteristics of bHLH proteins?

A
  • DNA-binding helices at N-termini of the monomers are separated by nonhelical loops from a leucine zipper-like region containing a coiled-coil dimerization domain.
  • They can form heterodimers.
31
Q

What is a distinct characteristic of DNA binding proteins?

A

work as dimers

32
Q

If you have three monomers that can form dimers, you can have ___ combonations

A

6

33
Q

What is an enhanceosome?

A

An enhanceosome is a protein complex that assembles at an enhancer region on DNA and helps to regulate the expression of a target gene

MCB 2050 (11 decks)

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