Transcription

Question 1

What are promoters?

Answer 1

The region of DNA that contains sequences that are signals for transcribing a gene.

Question 2

The promoter functions as a recognition site for transcription factors. The transcription factor (s) enable RNA polymerase to bind to the promoter. Following binding, the DNA is denatured into a bubble known as the open complex.

What stage of transcription is this?

Answer 2

Initiation

Question 3

RNA polymerase slides along the DNA in an open complex to synthesize RNA.

What stage of transcription is this?

Answer 3

Elongation/Synthesis of the RNA transcript

Question 4

A terminator is reached that causes RNA polymerase and the RNA transcript to dissociate from the DNA.

What stage of transcription is this?

Answer 4

Termination

Question 5

Name the 4 factors required for transcription in prokaryotes

Answer 5

1. RNA polymerase
2. Accessory transcription activator proteins
3. Template DNA containing gene or genes to be transcribed
4. Promoter

Question 6

What constitutes a core enzyme?

What are their functions?

Answer 6

3 different type of subunits:
☆B - binds incoming nucleotides
☆B’ - binds DNA
☆alpha- helps with enzyme assembly; interacts with other activator proteins; also interacts with some DNA sequences.

Question 7

What is the structure of a holoenzyme?

Answer 7

Core enzyme (2alpha units; B and B’) + sigma factor

Question 8

What is the function of a holoenzyme?

Answer 8

To recognize the promoter.

Question 9

What are the functions of accessory transcription activator proteins?

Answer 9

☆They bind to specific DNA sequences and help RNA polymerase initiate transcription via protein- protein interactions or by altering the structure of the DNA.

☆Some just increase the rate of transcription.

Question 10

What is the function of a promoter & where is it located?

Answer 10

☆It is the regulatory element that determines when a gene is “turned on” (transcribed) or “turned off”.
☆It is located upstream (towards the 5’ end) of the gene & contains a sequence which sigma factor of RNAP and other transcription factors can bind.

Question 11

What is a consensus sequence?

Answer 11

It is the sequence of nucleotides in DNA or amino acids in proteins most often present in a particular gene.

Question 12

What are weak promoters?

Answer 12

Promoters that have poor sigma recognition sequences & have additional sequences to which transcriptional activators can bind.

Question 13

How does transcription in eukaryotes differ from that of prokaryotes in terms of:

1. RNA polymerase
2. Arrangement of genes
3. mRNA being cistronic

Answer 13

Prokaryotes

1. RNA polymerase
   - RNA is synthesized by a single RNA polymerase & mRNA is translated during regulation.
2. Arrangement of genes
   - genes are contiguous (adjacent) segments of DNA, that are colinear with the mRNA that is translated into a protein.
3. mRNA being cistronic
   - is ployacistronic

Eukaryotes

1. RNA polymerase
   - 3 different polymerase are responsible for the different classes of RNA molecule.
2. Arrangement of genes
   - genes are often split, they are not contiguous segments of coding sequences, rather the coding sequences are interrupted by intervening sequences. Introns & Exons.
3. mRNA being cistronic
   - is monocistronic

Question 14

Discuss the functions of the three eukaryotic RNA polymerases.

Answer 14

RNA polymerase I :
- synthesizes rRna

RNA polymerase II :

• plays a major role in cellular transcription, because it transcribes all structural genes.
• synthesizes all mRNAs
• transcribes certain snRNA genes which are needed for pre-mRNA splicing.

RNA polymerase III
- trabscribes all tRNA genes and the 5S rRNA

Question 15

Where does transcription & translation in eukaryotes occur?

Answer 15

Transcription occurs in the nucleus.

Translation of the mRNA occurs in the cytoplasm.

Question 16

The initiation of RNA synthesis by RNA polymerase is directed by ….

Answer 16

The promoter site on the 5’side of the transcriptional start site.

Question 17

What is the name of the strand that is transcribed by RNA polymerase?

Answer 17

Antisense strand.

Question 18

Does RNA synthesis require a primer?

Answer 18

No.

Question 19

Where is the promoter site for RNA polymerase II located & what is the consensus sequence?

Answer 19

• It is located approximately 25-35 bp upstream of the start site.
• The start site is denoted as position +1, the TATA box is located at approximately position -25.
• TATA (A/T) A (A/T) , it is known as the TATA box.

Question 20

The TATA box sequence resembles the …

Answer 20

-10 sequence in prokaryotes TATAAT, except that it’s located further upstream.

Question 21

How is transcription regulated?

Answer 21

By upstream control elements that lie 5’ to the TATA box .

Question 22

What does RNA polymerase II require, in order to initiate transcription?

Answer 22

The help of several other proteins/protein complexes, known as general (basal) transcription factors.

TRANSCRIPTION FACTOR for RNA POLYMERASE II (TF II)

Question 23

What is the first event in initiation?

Answer 23

The binding of the transcription factor IID (TFIID) protein complex to the TATA box via one of its subunits called TBP (TATA box binding protein).

Question 24

What is the transcription initiation complex?

Answer 24

It is the final protein complex, that contains at least 40 polypeptides.

It is composed of:

TFIID bound to TBP + TFIIA which stabilizes the TFIID complex

TFIIB binds to TFIID, but TFIIB can also bind to RNA polymerase II

RNA polymerase II, is already complexed with TFIIF, also binds

It is followed by the binding of TFIIE & H.

Question 25

Which Transcription Factor for RNA polymerase II, acts as a bridging protein?

Answer 25

TFIIB

Question 26

What are the function of TFIIH?

Answer 26

1. It is a helicase, which means that it can use ATP to unwind the DNA helix, allowing transcription to begin.
2. It phosphorylates RNA polymerase II, which causes this enzyme to change its conformation & dissociate from other proteins in the initiation complex.

Question 27

Where does phosphorylation occur?

Answer 27

On a long C-terminal tail, called the C-terminal domain (CTD) of the RNA polymerase II molecule.

Question 28

What type of RNA polymerase II can initiate transcription?

Answer 28

Only one with a non-phosphorylated CTD.

Question 29

What type of RNA polymerase II can elongate RNA?

Answer 29

One with a phosphorylated CTD.

Question 30

RNA synthesis occurs in the 5’ to 3’ direction with the RNA polymerase catalyzing…

Answer 30

A nucleophilic attack by the 3-OH of the growing RNA chain on the alpha-phosphorus atom on an incoming ribonucleotide 5’-triphisphate.

Question 31

What is the RNA molecule made from a protein- coding gene by RNA polymerase II called?

Answer 31

A primary transcript.

Question 32

Elongation of the RNA chain continues until what occurs?

Answer 32

Termination

Question 33

Unlike RNA polymerase in prokaryotes, RNA polymerase II does not terminate transcription at a specific site, but rather…

Answer 33

Transcription can stop at varying distances downstream of the gene.

Question 34

What do RNA genes transcribed by RNA polymerase II lack?

Answer 34

Specific signals or sequences that direct RNA polymerase II to terminate at specific locations.

Question 35

What happens when the transcript is cleaved at an internal site before RNA polymerase II finishes transcribing?

Answer 35

An upstream portion of the transcript is released, which will serve as the initial RNA prior to further processing (the pre-mRNA)

Question 36

What is the cleavage site considered as?

Answer 36

The “end” of the gene.

Question 37

What happens to the remainder of the transcript?

Answer 37

It is digested by a 5’ - exonuclease ( called Xrn2 in humans), while it is still being transcribed by the RNA polymerase II.

Question 38

How is does the final round of termination of transcription occur?

Answer 38

The 5’- exonuclease “catches up “ to the RNA polymerase II by digesting away all the overhanging RNA, it helps disengage to polymerase from its DNA template.