Lecture 16

Question 1

Are transcription and translation coupled in prokaryotes? eukaryotes?

Answer 1

coupled in prokaryotes
uncoupled in eukaryotes because mRNA has to be transported outside the nucleus

Question 2

Compare/contrast euchromatin and heterochromatin

Answer 2

Euchromatin can be accessed by transcription/translation machinery because it’s relaxed
Heterochromatin is condensed and not transcriptionally active and does not have access to those regions of DNA

Question 3

Where is the region of heterochromatin?

Answer 3

close to the nuclear lamina

Question 4

What happens when genes need to be transcribed and become active?

Answer 4

decoiling… chromatin material extends to central point of nucleus so the transcriptional machinery can access it

Question 5

What is transcription mediated by?

Answer 5

sequence specific DNA binding transcription factors
the general RNAP 2 transcriptional machinery
coactivators and corepressors
elongation factors

Question 6

Does bacteria have enhancers and insulators? Core promoter?

Answer 6

no, and their core promoter is a lot more simplified in bacteria

Question 7

What is the TATA box? Do all core promoters have the TATA box?

Answer 7

specific DNA sequence found in the promoter region of genes that acts as a binding site for proteins marking the start point for transcription
No, only about 32% of core promoters have TATA box

Question 8

What is the typical structure of a gene in eukaryotes?

Answer 8

introns (spliced and not in the final mRNA sequence), exons (protein coding sequences), enhancers, core promoter (close to transition start site), and untranslated regions (not the whole transcript is going to be represented in a protein)

Question 9

Important facts about the core promoter?

Answer 9

required for initiation
increases the frequency of initiation only when near the +1 site
recognition site for RNAP 2 general transcription factors

Question 10

What are general transcription factors (or basal transcription factors)?

Answer 10

proteins that bind to specific promoter regions of DNA to initiate transcription by helping RNAP find and bind to the correct starting point of a gene

Question 11

What do enhancers do?

Answer 11

regulatory DNA sequences that increase the transcription of a gene by binding transcription factors

Question 12

Name an analogous (to the eukaryotic) general transcription factor in bacteria

Answer 12

sigma factor… helps bacterial RNAP locate and bind to the correct promoter regions on the DNA necessary to initiate transcription

Question 13

What is TFIID composed of?

Answer 13

TBP (TATA binding proteins) and TAF (TBP-associated factors)

Question 14

What do silencers do in transcription?

Answer 14

regulatory DNA sequences that suppress gene promoter activity or transcription of specific genes by binding repressor proteins which prevents transcription machinery from assembling or interferes with the promoter reducing or stopping gene expression

Question 15

What is an insulator? How do they work?

Answer 15

DNA sequences that make sure one part of the DNA remains separate from the other one, so they can’t influence each other
chromatin boundary marker
recognized by specific DNA binding proteins like CTCF

Insulators bind to insulator binding protein and make a loop which separates other parts of the DNA

Question 16

What is transvection and how does it work?

Answer 16

regulatory elements on one chromosome can influence the expression of a gene on its homologous chromosome
requires the homologous chromosomes to be closely paired which allows an enhancer from one chromosome to act on a promoter on the paired chromosome even across physical distance

Question 17

What is locust control region (LCR)?

Answer 17

region of DNA that controls the expression of a whole cluster of genes by maintaining an open chromatin structure

Question 18

What is topologically associating domain (TAD)?

Answer 18

discrete, self-interacting loop that often contains coregulated genes
co localize regulatory elements with target genes
act as insulators that prevent interactions between genes and regulatory sequences outside of the TAD

Question 19

How can you know if a putative enhancer is involved in gene expression regulation?

Answer 19

test DNA, clone it upstream of the reporter gene, transcribe construct into cells
reporter gene expression significantly elevated the enhancer is active

Question 20

What are reporter genes?

Answer 20

genetic tools to study how genes are regulated and expressed… they encode proteins with measurable and easily detectable product allowing researchers to track which cells have been successfully transfected

Question 21

Can the enhancer be developmental stage specific? How do we know?

Answer 21

Yes, because the blue tint caused by the enhancer moves around the embryo on different days

Question 22

How do GFP and Lac-Z help show the presence of enhancers?

Answer 22

GFP fluoresces green under UV light and Lac-Z is a gene that encodes a blue-producing enzyme, and both are used as reporter genes to indicate gene expression
Placed downstream of the enhancer sequence to a reporter construct and if the enhancer is active, it will drive the transcription of the reporter gene and its expression is visualized allowing researchers to see where and when a particular DNA sequence (the enhancer) is active in an organism

Question 23

What is X-gal?

Answer 23

a chromogenic substrate for the enzyme Beta galactosidase which is encoded by the Lac-Z gene so when cleaved by the B-galactosidase X-gal yields an insoluble, intensely blue precipitate used as a visual marker for gene expression