I. DNA & RNA | 11A. Structure and function of RNA polymerase of E. coli; initiation of transcription in prokaryotes; the prokaryotic transcription unit

Question 1

What are the 6 characteristics of transcription

Answer 1

1/ An RNA copy of DNA segments is made
2/ Sliding along the template strand follows 3’ to 5’ direction
3/ RNA chain grows in 5’ to 3’ direction
4/ Ribonucleoside triphosphate (NTPs) are used
5/ Starts without primer
6/ No proofreading

Question 2

Function of RNA polymerase

Answer 2

RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain

Question 3

Process of transcription

Answer 3

• The RNA moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing
• One of the two DNA strands serves as a template, and is copied in 3’-5’ direction
• The new RNA strand is elongated by adding ribonucleotides to the strand in 5’- 3’ direction

Question 4

Differences between RNA polymerase and DNA polymerase:

Answer 4

1. RNA polymerase catalyzes the linkage of ribonucleotides (not deoxyribonucleotides)
   meaning that all bases remain identical to those in the coding strand, except uracil replacing thymine
2. Unlike the DNA polymerases, RNA polymerases can start a RNA chain without the use of primer.
3. Unlike DNA polymerases, which make their products in segments that are later stitched together, RNA polymerases are absolutely possessive; that is, the same RNA polymerase that begins an RNA molecule must finish it without dissociating from the DNA template

Question 5

How is Direction of transcription along a short portion of a bacterial chromosome determined?

Answer 5

• Some genes are transcribed using one DNA strand as a template, while others are transcribed using the other DNA strand
• The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads)
• The genes transcribed from left-to-right use the bottom DNA strand as the template, while those transcribed from right-to-left use the top DNA strand as the template

Question 6

Structure of RNA polymerase:

Answer 6

The bacterial RNA polymerase is composed of
- 2 large subunits β and β’
- 2 smaller subunits α
- a σ-factor (sigma factor) that stabilizes the enzyme and assists in the assembly of the subunits

Question 7

What is Apoenzyme?

Answer 7

inactive form of enzyme that requires cofactor to be activated

Question 8

What is holoenzyme?

Answer 8

apoenzyme together with its cofactor

Question 9

Structure of RNA polymerase contain 5 subunits
-> What is the role of α subunits?

Answer 9

(1) responsible for assembly of the complex
(2) interaction with the promoter
(3) interactions with regulatory factors

Question 10

Structure of RNA polymerase contain 5 subunits
-> What is the role of β’ subunit?

Answer 10

forming catalytic center with β subunit + contains parts of the active center responsible for RNA synthesis

Question 11

Structure of RNA polymerase contain 5 subunits
-> What is the role of α subunits?

Answer 11

(1) responsible for assembly of the complex
(2) interaction with the promoter
(3) interactions with regulatory factors

Question 12

Structure of RNA polymerase contain 5 subunits
-> What is the role of β subunit?

Answer 12

forming catalytic center with β’ subunit + contains rest of the active center responsible for RNA synthesis

Question 13

Structure of RNA polymerase contain 5 subunits
-> What is the role of σ subunit?

Answer 13

the smallest subunit = binding to -10 and -15 sequences, ensuring promoter specificity to the complex.

Facilitates assembly of RNA polymerase and stabilizes assembled RNA polymerase

Question 14

Structure of RNA polymerase contain 5 subunits
-> What is the Role of σ-factor?

Answer 14

 the apoenzyme binds to any DNA sequence with high affinity
 general affinity of the holoenzyme to DNA is 1000 times lower
 at the same time, the holoenzyme has an increased affinity to promoter sequence

Question 15

What does Holoenzyme of RNA Polymerase of E.Coli contain?

Answer 15

(core + σ): α2ββ’σ
=> able to start synthesis at the right site (initiatio

Question 16

What does Apoenzyme of RNA Polymerase of E.Coli contain?

Answer 16

(core): α2ββ’
=> able to add nucleotide (elongation)

Question 17

How does RNA polymerase bind to DNA? (transcription unit)

Answer 17

Promoter sequences are DNA sequences that define where transcription of a gene by RNA polymerase begins

RNA polymerase (holoenzyme) binding occurs within a region stretching from about 70bp before the transcription start site to about 30bp beyond it

Promoters have similarities in 2 short sequences which are TATA box (at -10 region) and TTGACA (at -35 region)
=> These sequences are important interaction-site for the σ-factor

Question 18

How does initiation of transcription occur?

Answer 18

1/ Holoenzyme binds loosely to DNA and slides along until it finds a promoter
2/ Holoenzyme forms a “closed complex” with the promotor
3/ The tightly bound RNA polymerase (holoenzyme) opens up the double helix to expose a short stretch of nucleotides on each strand (‘’open complex’’ = in which the bound DNA is intact and partially unwound)
- The region of unpaired DNA (about 10 nucleotides) is called the transcription bubble and it is stabilized by the binding of σ-factor to the unpaired bases on one of the exposed strands.
- The other exposed DNA strand then acts as a template for complementary base-pairing with incoming ribonucleotides

4/ Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond
5/ Conformation changes again after about 10 nucleotides
=> Chain elongation is continued by the apoenzyme

Question 19

Structure of the prokaryotic transcription unit

Answer 19