post-transcriptional control of gene expression pt2

Question 1

What do ‘R’, ‘Y’, and ‘ψ’ represent in RNA?

Answer 1

R = Purine (G or A)
Y = Pyrimidine (C, T, or U)
ψ = Pseudouridine (most abundant posttranscriptionally modified nucleotide in cellular RNA)

Question 2

What is the function of aminoacyl-tRNA synthetases?

Answer 2

Aminoacyl-tRNA synthetases catalyze a 2-step reaction:
Amino acid activation: Amino acid + ATP bind to the catalytic site. The α-carboxylic acid oxygen undergoes nucleophilic attack, producing aminoacyl-adenylate (aa-AMP).
Aminoacylation of tRNA: The hydroxyl group of adenine 76 of tRNA attacks the carbonyl carbon of aa-AMP, forming aminoacyl-tRNA and releasing AMP.

Question 3

What happens during peptide bond formation in translation?

Answer 3

Peptide bond formation is catalyzed by the ribosome and involves:
A site: Aminoacylated tRNA binds.
P site: Growing polypeptide chain is attached to the tRNA.
E site: tRNA exits.
The reaction is a dehydration-condensation reaction.

Question 4

What is required for translation elongation?

Answer 4

Translation elongation requires:
Elongation Factor G (eEF2 in eukaryotes) and GTP for translocation.
Aminoacyl-tRNA binding at the A site.
Peptide bond formation between tRNAs in the P and A sites.
Translocation: The ribosome moves along the mRNA, shifting tRNAs through the A, P, and E sites.

Question 5

Key features of eukaryotic translation initiation

Answer 5

mRNA 5’ cap and poly-A tail are crucial for translation.
eIF4F complex (composed of eIF4E, eIF4G, and eIF4A) binds the 5’ cap, promoting mRNA circularization.
Small subunit of ribosome scans for the first AUG start codon.
Kozak consensus sequence (CC(A/G)CCAUG) helps identify the start codon in eukaryotic mRNAs.

Question 6

What is the role of eIF2 in translation initiation?

Answer 6

eIF2 binds Met-tRNA and GTP to form a ternary complex.
The eIF2-GTP complex is required for the initiation of translation at the AUG codon.
eIF2 is recycled via eIF2B, which is crucial for the initiation rate of translation.

Question 7

What regulates eIF2 activity?

Answer 7

eIF2 is regulated by phosphorylation, particularly on Ser51, which prevents its recycling by eIF2B.
Phosphorylation of eIF2 is triggered by stress conditions like:
Viral infection (PKR activation)
Amino acid deprivation (GCN2 activation)
Endoplasmic reticulum stress (PERK activation)

Question 8

What are the roles of IRP1 and IRP2 in translation regulation?

Answer 8

Iron Regulatory Proteins (IRP1 and IRP2) bind to Iron Response Elements (IREs) in the 5’ or 3’ UTRs of mRNAs that regulate iron metabolism.
Low iron levels cause IRP binding, blocking translation of iron import proteins or activating translation of iron storage proteins.
IRP1 has a bifunctional role: acting as c-aconitase in the absence of iron and as IRP in iron-deprived conditions.

Question 9

What is the role of the 5’ UTR and 3’ UTR in translation regulation?

Answer 9

Both the 5’ UTR and 3’ UTR regions can contain elements that influence translation efficiency, such as:
Iron Response Elements (IREs).
These regions interact with regulatory proteins like IRP1 and IRP2 to modulate translation and mRNA stability in response to cellular conditions, especially iron levels.

Question 10

What happens when the ribosome encounters a stop codon?

Answer 10

Translation stops when the ribosome reaches a stop codon.
Release factors recognize the stop codon and promote the release of the newly synthesized polypeptide chain.
The ribosome dissociates from the mRNA, ending translation.

Question 11

How does PKR contribute to the regulation of translation?

Answer 11

PKR is activated by double-stranded RNA (dsRNA), typically from viral infections.
PKR dimerizes upon binding dsRNA and autophosphorylates.
It then phosphorylates eIF2, preventing eIF2B from recycling eIF2, which reduces the initiation of translation in infected cells.

Question 12

What proteins and factors are involved in the formation of the 43S pre-initiation complex in eukaryotic translation?

Answer 12

The 43S pre-initiation complex involves:

eIF1, eIF1A: Aid in the recognition of the start codon.
eIF3: Promotes 40S subunit association and assists in the dissociation of 80S ribosome.
eIF2-GTP: Binds Met-tRNA and aids in its binding to the small ribosomal subunit.
Met-tRNA: Initiator tRNA carrying methionine.

Question 13

What role does eIF2-GTP play in the formation of the ternary complex during eukaryotic translation initiation?

Answer 13

eIF2-GTP forms a ternary complex with Met-tRNA, which is crucial for the assembly of the 43S pre-initiation complex. This complex is essential for the proper initiation of translation, guiding the small ribosomal subunit to the mRNA and facilitating its recognition of the start codon.

Question 14

How does the eIF4F complex facilitate mRNA circularization in eukaryotic translation?
A: eIF4F consists of eIF4E, eIF4G, and eIF4A:

Answer 14

eIF4E binds to the m7G cap of the mRNA.
eIF4G binds both eIF4E and other translation initiation factors like PABP, which binds the poly(A) tail of the mRNA.
eIF4A unwinds secondary structures in the mRNA’s 5’ UTR, facilitating scanning by the ribosome.
This complex helps circularize the mRNA, ensuring efficient translation initiation.

Question 15

What is the process of scanning in eukaryotic translation initiation?

Answer 15

The scanning process involves the small ribosomal subunit (as part of the 43S pre-initiation complex) binding to the m7G cap of the mRNA and moving along the 5’ UTR in search of the first AUG codon. The subunit scans until it encounters the start codon, where translation initiation occurs.

Question 16

What happens during translation termination in eukaryotes?

Answer 16

During termination, when the ribosome reaches a stop codon, release factors recognize the stop codon and catalyze the release of the newly synthesized polypeptide from the ribosome. The ribosome then dissociates into its subunits, and the mRNA is released, completing the translation process.

Question 17

How is eIF2B regulated and what role does it play in translation initiation?

Answer 17

eIF2B is a guanine nucleotide exchange factor (GEF) that catalyzes the exchange of GDP for GTP on eIF2, allowing the formation of the ternary complex essential for translation initiation.

eIF2B activity is downregulated in response to stresses such as viral infection, amino acid deprivation, and ER stress.
Phosphorylation of eIF2 (on Ser51) inhibits eIF2B, thus decreasing the availability of eIF2-GTP, reducing the initiation of translation.

Question 18

How does eIF2 phosphorylation regulate translation initiation?

Answer 18

Phosphorylation of eIF2 on Ser51 by various stress kinases (such as PKR, PERK, GCN2, and HRI) impairs its ability to exchange GDP for GTP. This prevents the formation of the ternary complex with Met-tRNA and reduces translation initiation. This mechanism helps cells respond to stresses like viral infections or nutrient deprivation.

Question 19

What are the main eIF2 kinases and what do they respond to?

Answer 19

Key eIF2 kinases include:

PKR: Activated by double-stranded RNA, often from viral infections.
PERK: Mediates the unfolded protein response during endoplasmic reticulum stress.
GCN2: Responds to amino acid deprivation, regulating general amino acid control.
HRI: Links iron availability to protein synthesis, especially in red blood cell biogenesis.
These kinases activate eIF2 phosphorylation, regulating translation initiation under stress conditions.

Question 20

How do iron levels regulate translation?

Answer 20

Iron levels influence the expression of iron-binding proteins, such as iron regulatory proteins (IRPs).

IRPs bind to Iron Response Elements (IREs) in mRNA’s 5’ or 3’ UTRs.
Binding of IRPs to IREs can either block or activate translation, depending on iron availability. When iron levels are low, IRPs bind to IREs to prevent translation of iron transport proteins, while iron binding can release these blockages.

Question 21

What are Iron Regulatory Proteins (IRPs) and how do they affect translation?

Answer 21

: IRPs are proteins that bind to Iron Response Elements (IREs) within the untranslated regions (UTRs) of iron-regulated mRNAs.

IRP1: Functions as both a regulatory protein and an enzyme involved in iron-sulfur cluster metabolism.
IRPs control the translation of iron metabolism proteins, affecting iron import and storage.
The binding of IRPs to mRNA can block translation of proteins involved in iron uptake when iron is abundant and activate translation when iron is low.

Question 22

What are Iron Response Elements (IREs), and where are they found?

Answer 22

IREs are hairpin loop structures found in the 5’ or 3’ UTRs of iron-regulated mRNAs. These regions have a conserved loop sequence and a bulge within the stem, and they are essential for the regulation of iron-related protein synthesis. The binding of Iron Regulatory Proteins (IRPs) to these elements influences mRNA stability and translation.

Question 23

What role does mRNA secondary structure play in translation initiation?

Answer 23

mRNA secondary structures, like hairpins in the 5’ UTR, can inhibit the ribosome’s ability to access the RBS or the start codon, affecting translation initiation.

Question 24

What are upstream open reading frames (uORFs) and their function in translation regulation?

Answer 24

uORFs in the 5’ UTR compete for ribosomal scanning or cause ribosome stalling, regulating translation of the main coding sequence by affecting translation initiation or reinitiation.

Question 25

How do RNA-binding proteins (RBPs) influence translation initiation?

Answer 25

RBPs bind to specific mRNA sequences and interact with initiation factors like eIF4E and eIF4G, either promoting or inhibiting the assembly of the translation initiation complex.

Question 26

What is the role of eEF1A in translation elongation?

Answer 26

eEF1A delivers aminoacylated tRNAs to the ribosome in a GTP-bound form. After GTP hydrolysis, the tRNA is released, and elongation continues.

Question 27

How does eEF2 contribute to translation elongation?

Answer 27

eEF2 catalyzes the translocation of the ribosome along the mRNA during elongation. It moves the ribosome one codon forward, allowing the next tRNA to enter.

Question 28

How do microRNAs (miRNAs) regulate translation?

Answer 28

miRNAs bind to the 3’ UTR of target mRNAs and inhibit translation initiation or cause mRNA degradation by guiding the RNA-induced silencing complex (RISC).

Question 29

What is the Shine-Dalgarno (SD) sequence in prokaryotic translation?

Answer 29

The Shine-Dalgarno sequence in bacterial mRNA pairs with the 16S rRNA of the small ribosomal subunit to facilitate ribosome binding and translation initiation.

Question 30

What role does fMet-tRNA play in prokaryotic translation initiation?

Answer 30

fMet-tRNA, carrying formylmethionine, binds to the start codon of bacterial mRNA during translation initiation, distinguishing it from eukaryotic methionine.

Question 31

What are the mechanisms for ribosome quality control?

Answer 31

Ribosome rescue mechanisms, like Pelota and Hbs1, dissociate stalled ribosomes from mRNA, and No-Go Decay (NGD) degrades mRNA that causes ribosome stalling.

Question 32

How do long non-coding RNAs (lncRNAs) regulate translation?

Answer 32

LncRNAs interact with mRNAs or translation machinery components, influencing translation by either promoting or inhibiting protein synthesis.

Question 33

What are the functions of small nucleolar RNAs (snoRNAs)?

Answer 33

snoRNAs guide the chemical modification of rRNAs, such as methylation or pseudouridylation, which is crucial for the proper assembly and function of ribosomes.

Question 34

How does eIF2 phosphorylation regulate translation?

Answer 34

Phosphorylation of eIF2 reduces its ability to bind GTP, inhibiting the formation of the initiation complex and thus suppressing translation initiation, especially under stress.

Question 35

What is the role of iron response elements (IREs) in translation regulation?

Answer 35

IREs in mRNA control the translation of genes involved in iron metabolism. Iron Regulatory Proteins (IRPs) bind to IREs, inhibiting translation when iron is low.

Question 36

Why does RNA undergo degradation?

Answer 36

RNA is degraded for various reasons, including the removal of damaged mRNA, incorrectly transcribed or processed mRNA, and to control gene expression.

Question 37

What is the half-life of RNA, and why is it important?

Answer 37

RNAs have a half-life ranging from minutes to days. This helps control gene expression by regulating the amount of mRNA available for translation.

Question 38

How is casein mRNA regulated during prolactin stimulation?

Answer 38

Casein mRNA levels increase ~70-fold in response to prolactin, even though transcription increases only ~2-fold. This increase is due to a dramatic increase in mRNA half-life and poly(A) tail length.

Question 39

What is the function of the circular structure of eukaryotic mRNAs during translation?

Answer 39

The circular structure helps monitor mRNA integrity. If the mRNA loses its cap or poly(A) tail, it cannot form a closed loop, which prevents translation and increases degradation.

Question 40

What happens during the first phase of mRNA degradation (deadenylation-dependent decay)?

Answer 40

mRNA loses its poly(A) tail, and enzymes like decapping enzymes, endonucleases, and deadenylases (e.g., DCP1, DCP2) initiate the breakdown of the RNA.

Question 41

What is the role of the exosome in mRNA degradation?

Answer 41

The exosome is the main 3’ to 5’ exonuclease in the cell, involved in RNA turnover and processing. It consists of multiple subunits with nuclease activities.

Question 42

How does XRN1 contribute to mRNA degradation?

Answer 42

XRN1 is a 5’ to 3’ exonuclease involved in RNA turnover and processing. It functions after the mRNA is decapped.

Question 43

What is Nonsense-Mediated Decay (NMD)?

Answer 43

NMD is a quality control mechanism that targets mRNAs with premature stop codons (PTCs) for degradation. It monitors mRNA structure, including splicing, polyadenylation, and editing.

Question 44

How is NMD triggered when a premature stop codon (PTC) is detected?

Answer 44

The Exon Junction Complex (EJC) marks the mRNA after splicing. When ribosomes encounter a PTC, an EJC remains downstream, attracting UPF proteins, which trigger mRNA degradation.

Question 45

How do miRNAs and siRNAs regulate gene expression?

Answer 45

miRNAs and siRNAs are small RNAs (21-23 nucleotides) that bind to target mRNAs. miRNAs regulate gene expression by blocking translation, while siRNAs target mRNAs for degradation.

Question 46

What is the role of the RNA-induced silencing complex (RISC)?

Answer 46

RISC is involved in both miRNA and siRNA pathways. It binds to small RNAs (miRNAs or siRNAs) and mediates the inhibition of translation or degradation of target mRNAs.

Question 47

How does the length of 3’ UTRs affect mRNA regulation?

Answer 47

During embryonic development, 3’ UTRs often become longer, providing more binding sites for miRNAs and regulatory proteins. In proliferating cells, 3’ UTRs are shorter.

Question 48

How are siRNAs used in research and clinical settings?

Answer 48

siRNAs are used in research to manipulate gene expression and in clinical applications, such as the treatment of diseases like hemophilia (e.g., fitusiran) and basal cell carcinoma (e.g., STP705).

Question 49

What are the key differences between miRNAs and siRNAs?

Answer 49

miRNAs: Have imperfect complementarity to target mRNA and block translation.
siRNAs: Have perfect complementarity to target mRNA and induce degradation.

Question 50

How do RNAi mechanisms contribute to gene regulation?

Answer 50

RNA interference (RNAi), through miRNAs and siRNAs, is a crucial gene regulatory mechanism, blocking translation or degrading mRNAs, thereby controlling gene expression.

Question 51

What are Exon Junction Complexes (EJCs), and where are they located?

Answer 51

EJCs are protein complexes located 20-24 nucleotides upstream of splice junctions. They are involved in mRNA surveillance and the detection of errors like premature stop codons (PTCs).

Question 52

How do mRNA levels vary despite constant transcription?

Answer 52

mRNA levels can vary dramatically due to turnover, where the degradation of mRNA changes its half-life and thus its concentration, even if transcription remains constant.

Question 53

What is the general process of mRNA degradation in eukaryotes?

Answer 53

mRNA degradation involves several steps: the mRNA is decapped, the poly(A) tail is removed, and exonucleases (such as XRN1 or the exosome) degrade the remaining RNA.

Question 54

What is the significance of the poly(A) tail in mRNA degradation?

Answer 54

The poly(A) tail is important for mRNA stability. Deadenylation, the process of shortening the poly(A) tail, triggers mRNA degradation by making it more susceptible to exonucleases.

Question 55

What is the role of the Ccr/Not complex in mRNA degradation?

Answer 55

The Ccr/Not complex is involved in the deadenylation-dependent decay pathway. It helps remove the poly(A) tail from mRNA, initiating the degradation process.

Question 56

What triggers the deadenylation-dependent decay pathway?

Answer 56

Deadenylation-dependent decay is triggered by a variety of mechanisms, including specific signals and interactions that lead to the gradual shortening of the poly(A) tail on mRNA.

Question 57

What are the key enzymes involved in the decapping phase of mRNA degradation?

Answer 57

The key enzymes involved in decapping include DCP1 and DCP2, which remove the 5’ cap structure from the mRNA, marking it for degradation.

Question 58

How do siRNAs and miRNAs differ in terms of their target interaction?

Answer 58

siRNAs: Bind perfectly to their target mRNAs, leading to mRNA cleavage and degradation.
miRNAs: Have imperfect complementarity to their target mRNAs and primarily inhibit translation without causing degradation.

Question 59

What are some clinical applications of siRNAs?

Answer 59

siRNAs have been used in clinical therapies such as fitusiran (for hemophilia) and STP705 (for basal cell carcinoma), where they silence specific genes to treat diseases.

Question 60

How does the length of the 3’ UTR affect the interaction with miRNAs?

Answer 60

A longer 3’ UTR provides more binding sites for miRNAs and regulatory proteins, which enhances the ability to regulate gene expression.

Question 61

What is the function of Argonaute proteins in RNAi mechanisms?

Answer 61

Argonaute proteins are key components of the RNA-induced silencing complex (RISC), which binds to small RNAs (siRNAs or miRNAs) and mediates the inhibition of translation or degradation of target mRNAs.

Question 62

What is the significance of the 5’ cap and poly(A) tail in mRNA stability?

Answer 62

The 5’ cap and poly(A) tail protect mRNA from degradation, but their removal is a critical step in the mRNA decay process, signaling that the mRNA should be degraded.

Question 63

What does the RISC complex do during miRNA and siRNA functions?

Answer 63

RISC, once bound to miRNAs or siRNAs, functions as a nuclease and inhibits translation or causes degradation of target mRNAs depending on the degree of complementarity.