52 Questions
Which enzyme is specifically inhibited by mushroom toxin a-amanitin?
RNA polymerase II
In which direction does elongation proceed during RNA synthesis?
5' to 3'
What is the function of the TATA box in eukaryotic RNA polymerase II promoters?
Is the major assembly point for proteins in preinitiation complexes
Which RNA polymerase is responsible for the synthesis of mRNA in eukaryotes?
RNA polymerase II
What is the significance of the Inr consensus sequence in transcription initiation?
Contains the transcription start site or is very near it
Which enzyme synthesizes tRNAs and some small RNA products in eukaryotes?
RNA polymerase III
What is the function of messenger RNAs in living cells?
Encode the amino acid sequences of all the polypeptides found in the cell
What is the main role of transfer RNAs in protein synthesis?
Match specific amino acids to triplet codons in mRNA during protein synthesis
What is the function of ribosomal RNAs in living cells?
Constitute the ribosomes and have structural and catalytic functions
Which type of RNA plays a role in regulating gene expression by binding to specific nucleotide sequences?
MicroRNA
What distinguishes long noncoding RNAs (lncRNAs) from other types of RNA molecules?
They do not code for proteins
What is the key function of Long noncoding RNAs (lncRNAs) in eukaryotic cells?
Contributing to cell differentiation and maintenance of cell identity
What is the general region where E.coli promoters occur to stimulate transcription?
Between -40 and -60
What is the purpose of radiolabeling the DNA fragment in protein-DNA footprinting experiments?
To visualize DNA bands on X-ray film
In the DNA footprinting experiment, what happens when DNA is bound by a protein like RNA polymerase at its binding site?
DNA is protected from chemical or enzymatic cleavage
What is the purpose of lane C in the experiment of RNA polymerase binding to the lac promoter?
Control for labeled DNA fragments
What role does the 5’ end of the nontemplate strand play in RNA polymerase binding experiments?
It is radioactively labeled for visualization
Which factor is responsible for unwinding DNA at the Inr region during transcription?
TFIIH
In which process is TFIIH involved besides transcription?
Repair
Which genetic diseases are associated with defects in TFIIH?
Xeroderma pigmentosum
What is the outcome of Pol II after termination?
Released, dephosphorylated, and recycled
How does TFIIH enhance transcription elongation?
Phosphorylation of Pol II's carboxyl-terminal domain
How does alpha-Amanitin from the mushroom Amanita phalloides selectively inhibit RNA polymerases?
By blocking Pol II and Pol III in eukaryotes
What is the mechanism of action of Rifampicin in inhibiting RNA polymerases?
Binding to the beta-subunit of bacterial RNA Pols
Which compound can work effectively on both prokaryotic and eukaryotic RNA polymerases?
Actinomycin D
How do Actinomycin D and Acridine inhibit DNA transcription?
They intercalate in DNA
What is the specific effect of a-Amanitin on RNA polymerases?
Blocking Pol II and Pol III in eukaryotes only
Why does a complex of actinomycin D with DNA lead to DNA bending?
Due to intercalation between G≡C base pairs in duplex DNA
Which compound specifically binds to the b-subunit of bacterial RNA Pols?
Rifampicin
What is the main difference between Rifampicin and a-Amanitin in terms of their target RNA polymerases?
Rifampicin targets bacterial RNA Pols while a-Amanitin targets eukaryotic RNA Pols
Actinomycin D and Acridine prevent transcription by binding to the major groove of the DNA double helix.
False
Rifampicin can selectively inhibit RNA polymerases in both prokaryotes and eukaryotes.
False
Alpha-Amanitin from the mushroom Amanita phalloides blocks Pol II of the mushroom itself.
False
Acridine inhibits DNA transcription by intercalating in DNA and bending it.
True
Actinomycin D binds to the minor groove of the DNA double helix and prevents DNA bending.
True
What happens during the initiation stage of RNA synthesis by RNA polymerase in E. coli?
The RNA polymerase searches DNA for initiation sites (promoters).
During elongation in RNA synthesis, which direction does the process proceed?
5' to 3'
What is a distinctive characteristic of RNA polymerase II in eukaryotes?
Its activity can be modified by activator and repressor proteins
What is the role of termination signals in the transcription process by RNA polymerase?
Signal the end of transcription
Which RNA polymerase in eukaryotes synthesizes pre-ribosomal RNA?
RNA Polymerase I
What is the major assembly point for preinitiation complexes of Pol II in eukaryotic transcription initiation?
TATA box
What is the primary function of the TATA box in eukaryotic transcription initiation?
Assemble preinitiation complexes
What is the rate of mRNA synthesis by RNA Polymerase II in eukaryotes (in nucleotides per second)?
500-1000 nucleotides/sec
What is the primary function of the ρ helicase in RNA transcription?
To migrate along the mRNA in the 5' to 3' direction and separate it from the polymerase
What is a characteristic feature of r-independent termination of transcription?
Formation of a hairpin at the 5' end
How is regulation achieved at the level of gene production during transcription?
By controlling the affinity of RNA polymerase for a promoter using different sequences and proteins
What happens to the σ subunit once elongation of RNA synthesis commences in E. coli?
It is released and replaced by the protein NusA.
What is the final step in transcription termination in E. coli after the RNA is released?
The RNA polymerase dissociates from the DNA.
During the initiation phase of transcription in E. coli, what are encompassed by this phase?
Transcription initiation and promoter clearance.
What leads to the formation of an open complex in transcription initiation in E. coli?
Unwinding of a 12 to 15 bp region from within the -10 region.
What happens when transcription is complete in E. coli after the RNA is released and NusA protein dissociates?
The RNA polymerase dissociates from the DNA.
What is the first step in the initiation phase of transcription in E. coli?
Formation of a closed complex
Study Notes
RNA Metabolism
- DNA-dependent synthesis of RNA occurs in three stages: initiation, elongation, and termination
- There are three main types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)
Transcription in E. coli
- RNA polymerase binds to specific DNA sequences called promoters
- The nucleoside triphosphates are added to the growing RNA chain in a complementary manner
- RNA synthesis occurs in the 5' to 3' direction
- RNA polymerase covers about a 35 bp-long segment of DNA
- Footprinting is used to analyze the binding of RNA polymerase to DNA
Transcription in Eukaryotes
- There are three main RNA polymerases in eukaryotes: RNA polymerase I, RNA polymerase II, and RNA polymerase III
- RNA polymerase II is responsible for synthesizing mRNA
- Eukaryotic transcription involves many proteins, including transcription factors, activators, and repressors
- RNA polymerase II has a carboxy-terminal domain that is phosphorylated during transcription
Initiation, Elongation, and Termination of Transcription
- Initiation: Assembly of RNA polymerase at the promoter, involving the TATA-binding protein (TBP) and other transcription factors
- Elongation: RNA polymerase continues to synthesize RNA, with the assistance of elongation factors
- Termination: RNA polymerase is dephosphorylated, and transcription is terminated
Regulation of Transcription
- Transcription is a major target for regulation in the cell
- Regulation can occur at the level of transcription initiation, elongation, and termination
- Activator and repressor proteins can bind to specific DNA sequences to regulate transcription
Inhibitors of Transcription
- Actinomycin D and Acridine: intercalate in DNA and prevent transcription
- Rifampicin: binds to the b-subunit of bacterial RNA polymerase and prevents transcription
- a-Amanitin: blocks RNA polymerase II and III in eukaryotes### Transcription Initiation and Elongation
- Transcription initiation and elongation in E. coli involves several steps:
- Binding phase: RNA polymerase binds to the promoter, forming a closed complex (DNA is not unwound)
- Initiation phase: a 12-15 bp region of DNA is unwound, forming an open complex
- Elongation phase: σ subunit is released, and NusA protein replaces it, allowing the polymerase to leave the promoter and commit to RNA elongation
- Termination phase: RNA is released, NusA protein dissociates, and RNA polymerase dissociates from the DNA
Transcription Termination in E. coli
- There are two types of transcription termination in E. coli:
- Rho-independent termination: characterized by a self-complementary region (palindrome) followed by 3 or more Us near the 3' end of the transcript, causing the RNA polymerase to pause and dissociate
- Rho-dependent termination: involves a rut site (Rho utilization element) and the Rho helicase, which separates the mRNA from the polymerase
Eukaryotic Transcription
- Eukaryotic mRNA transcription involves many proteins, including:
- RNA polymerase II: responsible for mRNA synthesis, with a large complex of 12 subunits
- TATA-binding protein (TBP): initiates transcription by binding to the promoter
- TFIIH: unwinds DNA at the promoter and phosphorylates the polymerase
- Elongation factors: enhance processivity and coordinate post-translational modifications
- Regulation of transcription is complex, involving many proteins interacting with different proteins of the transcription machinery
Promoters in Eukaryotic Transcription
- Promoters recognized by eukaryotic RNA polymerase II have:
- A TATA box: the major assembly point for preinitiation complexes
- An initiator sequence (Inr): where the DNA is unwound, and the transcription start site is usually located
- Additional sequences: recognized by one or more transcription factors, located within a few hundred base pairs of the TATA box or thousands of base pairs away
RNA Polymerase
- In E. coli, RNA polymerase:
- Searches for initiation sites (promoters)
- Unwinds a short stretch of dsDNA
- Selects correct NTPs and catalyzes phosphodiester bond formation
- Does not require a primer to initiate RNA synthesis
- Elongation proceeds in 5' to 3' direction
- Is completely processive and detects termination signals
- Its activity can be modified by activator and repressor proteins
Eukaryotic RNA Polymerases
- Eukaryotes contain several distinct RNA polymerases:
- RNA polymerase I: synthesizes pre-ribosomal RNA (precursor for 28S, 18S, and 5.8 rRNAs)
- RNA polymerase II: responsible for mRNA synthesis
- RNA polymerase III: makes tRNAs and some small RNA products
- RNA polymerase IV (in plants): responsible for the synthesis of small interfering RNAs
- Mitochondrial RNA polymerase: has its own RNA polymerase
Learn about the three stages of RNA synthesis in RNA Polymerase: Initiation, Elongation, and Termination. Understand how RNA Polymerase searches DNA for initiation sites, unwinds dsDNA, selects correct NTPs, and catalyzes phosphodiester bond formation.
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