Lecture 3 - Exam 2

Question 1

Telomerases are necessary in eukaryotes because…?

Answer 1

Eukaryotic DNA polymerases cannot copy all the way to the end of a chromosome.

Question 2

Describe the action of telomerase.

Answer 2

There is telomeric DNA with overhanging 3’ end. There is also a newly synthesized lagging strand. Then, binding to telomerase RNA occurs. Telomerase RNA binds to leading strand. Then, telomerase reverse transcriptase activity occurs. 3’ end of template strand elongated by one repeat unit.

Question 3

Bacteria: What is the promoter in the initiation step of transcription?

Answer 3

Gene sequence to which RNA polymerase binds to initiate transcription (in order to unwind DNA and start transcription).

Question 4

Bacteria: What does RNA polymerase do in the elongation step of transcription?

Answer 4

RNA polymerase catalyzes polymerization of ribonucleoside 5’-triphosphates (NTPs) as directed by a DNA template, in the 5’ to 3’ direction.

Question 5

What is the stop sequence in the termination step?

Answer 5

GC-rich sequence followed by seven A residues - only in bacteria.

Question 6

DNA is made up of?

Answer 6

Two polynucleotide chains. The bases are on the inside, joined by hydrogen bonds between complementary base pairs.
G with C
A with T

Question 7

What is a hydrogen bond?

Answer 7

Weak electrostatic attraction between a proton in one molecule and an electronegative atom in the other.

Question 8

Complementary base pairing allows for what?

Answer 8

Allows one strand of DNA (or RNA) to act as a template for synthesis of a complementary strand.

Question 9

Nucleic acids are capable of?

Answer 9

Self-replication

Question 10

RNA polymerase does what?

Answer 10

Catalyzes the polymerization of ribonucleotide triphosphates (rNTPs or NTPs) directed by a DNA template. Always 5’ to 3’.

Question 11

The sigma subunit identifies…?

Answer 11

The correct sites for transcription initiation.
It is weakly bound and can be separated from the other subunits.
Most bacteria have several different sigma factors that direct RNA polymerase to different start sites under different conditions.

Question 12

E. coli promoters are characterized by two sets of sequences (6 nucleotides long) located…?

Answer 12

10 and 35 vase pairs upstream of the transcription site (+1).

Question 13

The consensus sequences ______________ (at -10) and ___________ (at -35) are the bases most frequently found in different promoters.

Answer 13

TATAAT ;
TTGACA

Question 14

Mutations in the consensus sequences affect?

Answer 14

Promoter function.

Question 15

Describe transcription in bacteria.

Answer 15

Polymerase binds nonspecifically to DNA. Then, specific binding of sigma to -35 and -10 promoter sequences.
Initial binding: there is a closed-promoter complex. Unwinding of DNA around the initiation site… single stranded DNA is available. Now, there is an open-promoter complex. Initiation of transcription by joining of 2-3 ribonucleoside triphosphates (rNTPs).
After the addition of ~10 rNTPs the sigma subunit is released from the polymerase. Elongation of RNA occurs, and the polymerase maintains and unwound region of about 15 bp.
The B and B’ subunits form a crab-claw-like structure that grips the DNA template.
RNA synthesis continues until the polymerase encounters a stop signal. The newly made mRNA is released and the polymerase dissociates from its template. Alternatively, transcription of some genes is terminated by a specific termination protein (Rho), which binds extended segments of single-stranded RNA.

Question 16

Describe the bacteria stop sequence.

Answer 16

Transcription of the GC-rich inverted repeat results in a segment of RNA that forms a stable stem-loop structure. This disrupts its association with the DNA template and terminates transcription.

Question 17

What are the differences between prokaryotic and eukaryotic gene expression?

Answer 17

Eukaryotic cells have three RNA polymerases that transcribe different classes of genes. Eukaryotic RNA polymerases need to interact with a variety of additional proteins to specifically initiate and regulate transcription. Transcription takes place on chromatin; regulation of chromatin structure is important in regulating gene expression.

Question 18

What does a eukaryotic gene look like?

Answer 18

Promoter : binds RNA polymerase II as well as other proteins that help anchor RNA pol II to the promoter.
Transcription start site : AKA the 5’ cap o f the resulting mRNA molecule.
5’ UTR : leader sequence; helps with rate of transcription/translation.
Exons and introns present.
3’ UTR : transcribed but not translated. Poly A addition site, which is critical for protein translation.

Question 19

Describe Eukaryotic RNA polymerases.

Answer 19

Complex enzymes, with 12 to 17 different subunits. They all have nine conserved subunits, five of which are related to subunits of bacterial RNA polymerase. Yeast RNA polymerase II is strikingly similar to that of bacteria.

Question 20

What is different between a prokaryotic promoter and an eukaryotic promoter?

Answer 20

Prokaryotic promoter: -35 and -10 sequences upstream from +1 transcription start site.
Eukaryotic promoter: -100 sequence (GGGCGG), -75 (CCAAT), -50 (GGGCGG) and -25 (TATAA) upstream of +1 transcription start site.

Question 21

In eukaryotic promoters, promoters (TATA and GC boxes), were identified in…?

Answer 21

Studies of the herpes simplex virus gene that encodes thymidine kinase.

Question 22

Is there a sigma subunit present in eukaryotic RNA polymerases?

Answer 22

NO!

Question 23

What are transcription factors?

Answer 23

Are regulatory proteins whose functions is to activate (or inhibit) transcription of DNA by binding to specific DNA sequences.

Question 24

Transcription factors have defined DNA-binding domains with up to…?

Answer 24

106-fold higher affinity for their target sequences that for the remainder of the DNA strand.

Question 25

What RNA polymerase is used in initiation of transcription in eukaryotes?

Answer 25

RNA pol II

Question 26

Eukaryotic transcription: General transcription factor, _________, (with 15 subunits) binds to the DNA and is followed by the recruitment of a second general transcription factor _________.

Answer 26

TFIID ;
TFIIB

Question 27

What is TBP?

Answer 27

TATA-binding protein

Question 28

What is TAF?

Answer 28

TBP associated factors

Question 29

Eukaryotic transcription: After TFIIB binds to TFIID, TFIIB will serve as a….?
After this, is the polymerase complex moving?

Answer 29

TFIIB in turn serves as a bridge to RNA polymerase II, which binds to TBP and a third factor TFIIF.
STILL NOT MOVING YET!!!

Question 30

TFIIB is serving as a bridge to RNA pol II, which binds to TBP and TFIIF. What happens after this?
Is the complex able to move along the DNA now?

Answer 30

TFIIE and TFIIH bind next.
STILL NOT ABLE TO GO.

Question 31

What is the role of TFIIH?

Answer 31

Two roles:
1) Helicase that unwinds DNA
2) Protein kinase that phosphorylates the C-terminal domain (or CTD).

Question 32

After TFIIE and TFIIH bind, what happens?

Answer 32

Finally, the mediator complex binds.
The mediator complex is 20 subunits, which stimulates BASAL transcription while also linking the general TFs to the gene specific TFs that regulate transcriptional activity.

Question 33

Eukaryotic transcription: When can the complex start moving along the DNA and initiate transcription?

Answer 33

Transcription initiates following phosphorylation of the CTD by TFIIH. Phosphorylation of the CTD happens after the mediator complex binds, then initiation of transcription occurs,

Question 34

What are enhancers?

Answer 34

Positive cis-regulatory elements that are regulatory sequences located farther away from the start site (upstream). They help recruit and stabilize RNA polymerase on specific promoters.

Question 35

Without an enhancer, what happens?

Answer 35

The gene is transcribed at a low basal level. Addition of an enhancer, stimulates transcription.

Question 36

Does the activity of enhancers depend on their distance from, or orientation with respect to the transcription initiation site?

Answer 36

It does not depend on their distance from, or orientation with respect to the transcription initiation site.

Question 37

What are the transcription factor (TF) domains?

Answer 37

DNA binding domain, protein-protein interaction domain, and the transactivating domain.

Question 38

What is the DNA binding domain?

Answer 38

Different TF families bind DNA differently.

Question 39

What is the protein-protein interaction domain?

Answer 39

Used to form homo- or heterodimers.

Question 40

What is the transactivating domain?

Answer 40

Interacts with RNA pol II or co-activating or co-repressor proteins that can modify histones to either de-condense or condense chromatin among other things.

Question 41

Gene expression is also regulated by repressors, which..?
In some cases, they simply…?
Other repressors….?

Answer 41

Inhibit transcription.
In some cases, they simply interfere with binding of other transcription factors.
Other repressors compete with activators for binding to specific regulatory sequences.

Question 42

Does RNA pol II sit on every promoter?

Answer 42

NO!

Question 43

The TATA box (where RNA pol II binds) is close to the _____________.

Answer 43

Transcription start site.

Question 44

Usually, upstream promoter elements (enhancers) can be quite distant from the transcription start site, often _______ bps away and as much as a ________ bps away.

Answer 44

20,000 ; million

Question 45

What binds promoters to enhancers?

Answer 45

TFs can form bridges so that the enhancers and the modifying proteins are brought to the promoter region. (In the picture on the slides, it shows the DNA bending over itself, and a bridge forming).

Question 46

KEY CONCEPT: Promoter + Enhancer = ?

Answer 46

Cis-regulatory element (CRE)

Question 47

In eukaryotes, __________ are extensively modified before export from the nucleus.

Answer 47

Pre-mRNAs