Transcription in Prokaryotes - week 3

Question 1

what is transcription

Answer 1

Transcription is a process by which a molecule of RNA is formed from a double-strand DNA template, which means it is the synthesis of an RNA molecule using a DNA template.

Question 2

which direction does RNA synthesis occur

Answer 2

RNA synthesis occurs in 1 direction, which is the 5’ > 3’ direction

Question 3

which enzyme is responsible for synthesis RNA molecule

Answer 3

RNA polymerase is the enzyme responsible for transcription, essentially RNA polymerase is the enzyme that synthesises RNA molecules.
RNA polymerases are usually complex multi-subunit enzymes.

Question 4

what is the template strand and sense-strand

Answer 4

Only 1 strand of the DNA template is transcribed. The sequence of this DNA is called the template strand.
The sequence of the newly generated strand is complementary to the template strand and it is the same as the coding strand also called the sense strand.

Question 5

what is gene expression

Answer 5

Gene expression is the process overall by which the genetic information within a gene is transformed or is used to form a product that is the protein or the polypeptide.
Transcription is the first stage in gene expression (Transcription occurs in the nucleus) and it leads to the synthesis of a protein

Question 6

what are the different types of RNA and where are they involved

Answer 6

the different types of RNAs are:
- tRNA
- rRNA
- snRNA
- mRNA
Not all the RNAs produced by transcription are translated into proteins. Only the mRNA is translated into proteins. tRNA and rRNA are involved in translation and snRNA is involved in mRNA processing in eukaryotes.

Question 7

what strand is complementary to RNA and which strand is identical to RNA

Answer 7

The coding strand sequence will be IDENTICAL to the RNA
Template strand will be COMPLEMENTARY to RNA

Question 8

what enzyme catalyses the transcription

Answer 8

Transcription is catalysed by RNA polymerase

Question 9

where does RNA polymerase bind to DNA

Answer 9

RNA polymerases bind to DNA at specific sites called promoters. RNA polymerase binds to dsDNA either alone or in association with accessory factors that initiate transcription called transcription factors

Question 10

what are the 3 stages transcription occurs in

Answer 10

Transcription occurs in 3 stages:
- initiation
- elongation
- termination

Question 11

what are the 2 components that make up E.coli polymerase and what is their function

Answer 11

The E.coli RNA polymerase is a complex molecule of six subunits grouped into two components:
- Core enzyme (2α, β, β’, ω) will begin transcription just about anywhere. It is responsible for transcription elongation
- Sigma factor (σ) provides specificity and is primarily responsible for promoter recognition. The most common σ subunit in E.coli is σ70

The entire collection of subunits is called RNA polymerase (Holoenzyme).

Question 12

why are the 2 alpha subunits in E.coli RNA polymerase important

Answer 12

The 2 alpha-subunits are important for the formation of the holoenzyme and are also important in the process of binding to the DNA.

Question 13

why are the 2 beta-subunits in E.coli RNA polymerase important

Answer 13

The 2 beta-subunit are also important for binding to the DNA but they are specifically important for the catalytic function, so they carry out the catalytic synthesis of RNA.

Question 14

why is the omega subunit in E.coli RNA polymerase important

Answer 14

The omega subunit is important for the proper assembly of the core enzyme and it also has some important regulatory functions.

Question 15

why is the sigma- subunit in E.coli RNA polymerase important

Answer 15

The sigma-subunit, which in E.coli is sigma-70, is important for the specificity of RNA holoenzyme to the DNA, and to recognise the promoter.
The promoter is the specific site within the DNA sequence where the transcription starts.

Question 16

what happens in the initiation step briefly in transcription

Answer 16

in short, Initiation is when the RNA polymerase binds to the DNA at a specific site and opens up the DNA, so essentially RNA polymerase unwinds the DNA template to form a bubble of unpaired DNA and in this bubble transcription process occurs. RNA polymerase unwinds the DNA template to form a bubble of unpaired DNA and in this bubble, the transcription process occurs.

Question 17

what happens in the elongation step briefly of transcription

Answer 17

The elongation step essentially is the step of transcription in which the RNA polymerase synthesises the RNA.

Question 18

what is the termination step briefly of transcription

Answer 18

The termination is the final step of the transcription process in which the RNA transcript and the RNA polymerase are released from the DNA template and the bubble closes.

Question 19

what is a promoter and what is its function

Answer 19

Promoters are sequences in the DNA just upstream of the RNA synthesis initiation site. They contain conserved (consensus) sequences required for specific binding of RNA polymerase.
The role of the promoter is to attract RNA polymerase to the correct start site so transcription can be initiated

Question 20

what are the 2 important regions in the promoter

Answer 20

There are 2 important regions within the promoter that are important for transcription, which are:
- -35 region
- -10 region

Question 21

why are the 2 important regions within a promoter (-35 and -10 region) negative numbers

Answer 21

The first nucleotide that is transcribed during transcription is indicated as +1, and everything upstream of the first nucleotide is indicated as a negative direction (number), and the promoter is a specific sequence upstream of the initiation site +1, and it consists of 2 important regions, one at -35 region and the other at -10 region.

Question 22

why is the -35 region important

Answer 22

-35 Sequence (which has 6bp elements): TTGACA is highly conserved in the most efficient promoters. It enhances the binding of the sigma factor of RNA polymerase.

Question 23

why is the -10 region important

Answer 23

-10 Pribnow box/-10 region (which has 6 bp elements) is the TATAAT-rich region and it is important for DNA denaturation which is important for the initiation of transcription.

Question 24

why do core enzymes bind to the enzyme anywhere

Answer 24

The core enzyme binds to the DNA anywhere because the core enzyme does not recognise a specific sequence within the DNA template.

Question 25

what happens in DETAIL in 5 steps at the initiation/elongation steps of transcription

Answer 25

Step 1: At the beginning, the sigma factor within the holoenzyme binds to the promoter, it recognises the promoter, which is located upstream of the initiation site, and following the promoter binding, there is a formation of the closed complex.
step 2: Then the 17bp of the DNA is unwound by RNA polymerase forming an open complex which is a transcription bubble
Step 3: Then the RNA polymerase starts to synthesise very short RNA molecules, (RNA polymerase is able to start the polymerisation of RNA molecules without the need for a primer which is something that DNA polymerase is not able to do).
Step 4: After the RNA polymerase synthesises very short RNA molecules, the sigma factor is released and the core enzyme can proceed and can continue to synthesise RNA. This is because in the presence of the sigma factor, the enzyme that RNA polymerase binds to the template very tightly, so it’s not possible to move thus the sigma factor is released and the core enzyme can continue the transcription.
Step 5: When the sigma factor is released/ the dissociation of the sigma factor from the RNA polymerase marks the transcription to the elongation step where the RNA polymerase continues to add nucleotides to the RNA chain.

Question 26

what happens in DETAIL in the termination step of transcription

Answer 26

The termination is the final step of transcription.
RNA polymerase continues to transcribe the DNA until it reaches a specific sequence that is called terminator sequence, so when the RNA polymerase reaches the stop signal, the RNA polymerase dissociates from the DNA template and also the RNA transcript also dissociates from the DNA template.

Question 27

what are the 2 different mechanisms in E.coli

Answer 27

In E.coli, 2 different mechanisms for termination have been identified, which are:
1. Rho independent
2. Rho dependent

Question 28

what is Rho

Answer 28

Rho is a protein and it is involved in the termination step of transcription in E.coli

Question 29

how does the Rho-independent process occur in DETAIL in 5 steps

Answer 29

Step 1: The RNA polymerase reaches a specific sequence within the RNA, this specific sequence is called Inverted Repeat (IR).
Step 2: These sequences are transcribed, due to their particular sequence being rich in GC, they form a stem loop.
Step 3: The RNA polymerase, after the formation of the stem-loop, pauses (it stops) in the synthesis of RNA Step 4: During this pause, the Uracil-rich sequence in the newly synthesised RNA molecule is bound to the DNA template because the hydrogen bonds between U and A are weaker than A and T,
Step 5: The transcript is released from the RNA polymerase and the DNA template.

Question 30

what are some facts about Rho independent mechanism

Answer 30

The process of Rho-independent termination does not require a protein.
Another characteristic of the Rho-independent mechanism is the presence of an A-rich region downstream of the inverted repeat sequence. This AAAA-rich region after the transcription is transcribed in the uracil-rich region.

Question 31

how does the Rho dependent process occur in DETAILin 5 steps

Answer 31

Step 1: When RNA polymerase transcribes the RNA, the Rho factor binds to the RUT site on the transcript and walks along the transcript in the direction of the RNA polymerase.
Step 2: When the RNA polymerase reaches the IR (inverted repeat) sequence, there is the formation of the stem loop, which causes the RNA polymerase to pause.
Step 3: Then the Rho protein catches up to the RNA polymerase and functions as a helicase because it is able to unwind the DNA/RNA hybrid base pairs.
Step 4: After this, all of the components, RNA polymerase, RNA transcript, and Rho protein are released from the template
Step 5: transcription is terminated.

Question 32

what are some facts about Rho protein

Answer 32

• Rho protein is an essential protein in bacteria such as E.coli.
• Rho protein consists of 6 subunits, it is an ATP-dependent protein and it functions as a Helicase because it is able to separate the hybrid of DNA/RNA.
• This Rho protein binds to a specific sequence called RUT ( Rho utilisation site).