Transcription

Question 1

What is the role of mRNA.

Answer 1

mRNA acts as the template for protein synthesis.
It is the product of gene transcription.

Question 2

where does translation take place?

Answer 2

Prokaryotes (bacteria and archaea) –> in the cytoplasm (they don’t have a nucleus)
Eukaryotes –> in the nucleus

Question 3

what are the different kinds of mRNA in PROKARYOTES.

Answer 3

Prokaryotic mRNA can be polycistronic or monocistronic:

*Polycistronic – one mRNA which encodes for several different proteins.

*Monocistronic - one mRNA which encodes for just one protein.

EUKARYOTIC mRNA= monocistronic

Question 4

what is an operon.

Answer 4

An operon is a functional unit of DNA containing a cluster of related genes under the control of a single promoter. These genes are transcribed together into a single mRNA molecule.

Question 5

what are the components of An operon?

Answer 5

Promoter: where RNA polymerase binds to initiate transcription.

Operator: A regulatory sequence that can interact with repressor or activator proteins to control gene expression.

Structural Genes: The actual genes that encode proteins, often related in function.

Question 6

do eukaryotes have operons?

Answer 6

No, only prokaryotic operons.
inastead Each gene has its own promoter and regulatory elements (rather than a cluster).

Question 7

what do monocistronic genes have instead of operators to control their transcription?

Answer 7

monocistronic genes can have various regulatory sequences (such as enhancers, silencers, and promoters, ORF)

Question 8

What are the regulatory elements that control gene expression in eukaryotic cells?

Answer 8

open reading frame (ORF)
Promoter
enhancer
silencer

Question 9

Open reading frame (ORF)

Answer 9

Open reading frame (OFR):
The DNA sequence that encodes for a protein, starting from a start codon and ending at a stop codon.

Question 10

Promoter

Answer 10

A promoter is a specific sequence of DNA where transcription proteins, including RNA polymerase and transcription factors, bind to initiate the process of RNA transcription.

Essential for the regulation of gene expression, determining when and how much a gene is transcribed.

Question 11

Enhancer

Answer 11

A DNA sequence that can bind ‘activator’ proteins to increase the likelihood of transcription.
Enhancers can be located anywhere within the genome they regulate and can act in any orientation, facilitating the recruitment of transcription machinery to the promoter.

Question 12

Silencer

Answer 12

A DNA sequence where ‘repressor’ proteins bind to inhibit/silence transcription.
Silencers can also be located at various distances from the gene, preventing or decreasing gene expression.

Question 13

What are the three stages of transcription?

Answer 13

Initiation
Elongation
Termination

Question 14

What is required for the Transcription initiation in PROKARYOTES.

Answer 14

• core RNA polymerase- This enzyme is essential for synthesizing RNA.
• requires σ (sigma) factor- To recognise and bind with promoter sequences.
• Promoter Region: The DNA must have a suitable promoter region where the RNA polymerase-sigma complex can bind.
• For transcription to start, the σ factor needs to be released allowing the core RNA polymerase to continue elongating the RNA strand without the sigma factor

Question 15

what happens in the Transcription initiation phase in PROKARYOTES?

Answer 15

Formation of the RNA Polymerase-Sigma Complex:
- The core RNA polymerase binds to the sigma factor (σ), forming a holoenzyme.

Binding to the Promoter:
- The RNA polymerase-sigma complex binds to the promoter sequence.

DNA Unwinding:
- Once bound to the promoter, the DNA strands unwind, creating a transcription bubble exposing a template strand for RNA synthesis.

RNA Synthesis Begins:
- The RNA polymerase starts synthesizing a short RNA strand by catalyzing the formation of phosphodiester bonds between nucleotides.

Release of the Sigma Factor:
After synthesizing about 10-12 nucleotides, the sigma factor is released.

Transition to Elongation:
The polymerase transitions into the elongation phase, where it continues to add nucleotides to the growing RNA chain.

Question 16

Transcription initiation in EUKARYOTES.

Answer 16

is more complex than in prokaryotes- a variety of transcription factors (TFs)/General Transcription Factors (GTFs) are needed to help recruit RNA polymerase II to the promoter.

The order in which thye come =DABFEH - Dont Always Believe Foxes Eat Helens

TFIID:
TFIIA:
TFIIB:
TFIIF:
TFIIE:
TFIIH:

Question 17

What is serine 5

Answer 17

The fifth amino acid on the RNA polymerase which is phosphorylated becomes (Ser5P)-
*P stands for phosphorylated.

Question 18

What happens in the Initiation phase of transcription in EUKARYOTES?

Answer 18

TFIID: Contains TBP and TAFs, binds to the TATA box in the DNA promoter region.

TFIIA: Joins TFIID, stabilizes the binding, and prevents any repressor proteins.

TFIIB then binds to the TFIID-DNA complex. TFIIB acts as a bridge between TFIID and RNA polymerase II, positioning RNA Pol II correctly at the promoter.

TFIIF then joins the complex, stabilizing the interaction between TFIIB and RNA polymerase II.

Following this, TFIIE helps recruit TFIIH to the complex and plays a role its formation

Finally, TFIIH is recruited to the complex.
TFIIH has dual roles:
- it possesses helicase activity that unwinds the DNA, allowing access to the template strand.
- phosphorylates serine 5 of the C-terminal domain (CTD) of RNA polymerase II- this facilitates the transition from initiation to elongation, as it marks RNA Pol II is ready to move beyond the promoter region and begin elongating the RNA transcript.

Question 19

what is the promoter Clearance.

Answer 19

Once RNA pol II is phosphorylated, it can start reading the DNA template strand, synthesizing RNA, and moving away from transcription factors and the promoter region and begins the elongation phase. This process is called promoter clearance,.

Question 20

What else does the phosphorylation of Serine 5 on RNA Pol II do ?

Answer 20

The phosphorylation of serine 5 not only facilitates transcription but also plays a key role in mRNA processing:
- helps attract/recruit a capping enzyme to the growing RNA strand.

• The capping enzyme adds a 5’ cap to the end of the newly synthesized mRNA.

Functions of the 5’ Cap:

Protection: The cap protects the RNA from degradation by exonucleases.

Nuclear Export: It aids in the transport of the mRNA from the nucleus to the cytoplasm.

Translation: The 5’ cap is crucial for the initiation of translation, helping ribosomes recognize the mRNA.

Question 21

Transcription Elongation in prokaryotes.

Answer 21

The RNA polymerase moves along the DNA template strand, synthesizing RNA in the 5’ to 3’ direction by adding complementary RNA nucleotides to the DNA template.

This newly emerging RNA is called nascent RNA. The process continues until RNA polymerase reaches a termination signal, completing transcription.

Question 22

Transcription Elongation in Eukaryotes.

Answer 22

RNA Polymerase II gets phosphorylated at specific serine residues on its C-terminal domain (CTD).

Initially, Serine 5 is phosphorylated, promoting the addition of the 5’ cap to the nascent RNA.

Later, Serine 2 is phosphorylated, which helps recruit factors for RNA splicing, 3’ end processing, and polyadenylation.

These phosphorylation events coordinate transcription with RNA processing, ensuring the RNA is properly modified as it is synthesized.

Question 23

Transcription termination in prokaryotes.

Answer 23

Well characterised

Rapid and efficient termination

Rho-independent (50% in E. coli)

Rho-dependent (50% in E. coli)

Question 24

Rho independent:

Answer 24

A hairpin loop is created in the RNA and folds within the RNA Polymerase.

Along with the hairpin loop, the area of
weak binding at the poly-A site in this
template sequence, leads to dissociation of the RNA from the DNA = termination.

Question 25

Rho dependent

Answer 25

Rho (ρ) factor required = RNA-binding ATP-dependent helicase.

Rho factor binds to rho utilisation site (rut)
on the RNA transcript.
ATP hydrolysis translates Rho along the
transcript towards RNA polymerase.
Rho reaches the DNA-RNA heteroduplex
and dislodges the RNA from the DNA
template, causing termination.

Question 26

Transcription termination in EUKARYOTES.

Answer 26

• Triggered by the polyadenylation signal 5’AAUAAA3’ on the RNA.
• RNA is cleaved 10-30 nucleotides downstream by the polyadenylation complex.
• RNA polymerase falls off the template DNA – involves the polyadenylation complex.