2/3 DNA to Protein: Transcription and Translation

Question 1

What are microRNA (miRNA) and what are the functions?

Answer 1

• small non-coding RNA molecule (~22 nucleotides) that function in RNA silancing and post-transcriptional regulation of gene expression.
• Gene silancing in plants and animals

Question 2

What are the steps for microRNA biogenesis?

Answer 2

1. pre-miRNA folds into particular structure w/ hairpin loop
2. pre-miRNA gets trimmed (microprocessor formation)
3. Nucleus → cytoplasm
4. Dicer cuts off hairpin loop → miRNA duplex (miRNA + mRNA)
5. Dicer (RISC coplex) further trims miRNA and pairs it with mRNA transcript

• *miRNA needs to be single stranded to be activated

Question 3

Can miRNA’s target mutliple genes?

Answer 3

• many miRNAs can target one gene or….
• one miRNA can target many genes

Question 4

What does miRBASE archieve?

Answer 4

• information about miRNAs including clinical significance

Question 5

How are rRNAs modified in the nucleolus before they leave the nucleus?

Answer 5

• 2’ O methylated nucleotide
• pseudouridine (rotation of side chains)
• Ribosomal RNAs have more than 100 of each type of modification
• snoRNAs determine the sits of modification by base-pairing to complementary sequences on the precursor rRNA. The snoRNAs are bound to proteins, and the complexes are called snRNPs.
  
  • SnoRNPs contain the guide sequences and the enzumtes that modify the rRNA.

Question 6

What happens in the nucleolus?

Answer 6

1. Assembly of the ribosomal complex
2. Assembly of the telomerase complex

• its not membrane bound.
• pre and post processing - rRNA and the proteins needed to asssemble the ribosome.

Question 7

How does the Nucleolus produce Ribosomes?

Answer 7

• Precursor rRNA is packaged with many proteins
• → Immature large & small ribosomal subunits
• Final processing doesn’t occur until the immature formsare shipped through the nuclear pore to the cytoplasm.

Question 8

What happends at the location of cajal bodies?

Answer 8

• Cajal bodies are where most snoRNAs are assembled into snRNPs

Question 9

What is the tRNA structure like?

Where is the acceptor stem & anticodon within the structure?

Answer 9

Question 10

What is the wobble position?

Answer 10

• Some tRNAsonly requrie accurate base-pairingonly at the first two positions of the codon and can tolerate a mismatch (or wobble) at the third position.
• Wobble position makes it possible to fit the 20 amino acids to their 61 codons with as few as 31 kinds of tRNA molecules.

Question 11

Why do tRNAs splice introns out by a specialized set of proteins and not the spliceosome?

Answer 11

• Trimming and splicing require the tRNA to be folded properly and will not proceed if the tRNA is misfolded.
• The splicesome is not set up to take into account 3D structure
• So specialized proteins evolved just for tRNAs

Question 12

What happens before tRNAs exit the nucleus?

Answer 12

• tRNAs are covalently modified before they exit from the nucleus.
• ~10% of tRNAs are modified.

Question 13

What is aminoactyl-tRNA synthetase?

Answer 13

• aminoactyl-tRNA synthetase helps ensure that the correct amino acid is coupled to the tRNA
• The tRNA is an adapter whose anticodon pairs with the codon in the mRNA to identify the correct amino acids to add to the growing peptide.
• 3D interaction between amino acids of protein and nucleotides of tRNA.
  *

Question 14

What is the fundamental reaction of protein synthesis?

Answer 14

• the formation of a peptide bond between the carboxyl group at the end of a growing peptide and the amino group on the incoming AA.
• Formation of the peptide bond is energetically favored.

Question 15

Prokaryotic vs Eukaryotic ribosomes

Answer 15

• Bacterial Ribosomes
  
  • 70S
    
    • 50S, 30S
• Eukaryotic Ribosomes
  
  • 80S
    
    • 60S, 40S

Question 16

What binding sites does a ribosome have?

Answer 16

• each ribosome has one binding site for mRNA and 3 sites for tRNA
• A, P and E.
• A= amino actl tRNA
• P= peptidyl-tRNA site
• E= exit

Question 17

What are the four steps in protein synthesis?

Answer 17

1. New tRNA binds to A site pairing with codon
2. Carboxyl end of growing peptide is released from tRNA in P site. Peptide bond formation between the previous AA added and the new one. tRNAs are in P site and A site
3. Large subunit moves along mRNA held by small subunit, shifting tRNAs to P and E sites on small subunit
4. Small subunit moves 3 nucleotides along the mRNA and ejects the tRNA in the E site.

Question 18

What do Elongation facotrs do in protein synthesis?

Answer 18

• elongation factors drive translation forward and improve its accuracy

Question 19

What is the primary role of proteins?

Answer 19

• to stabilize the RNA core.

Question 20

Is Ribosome a Ribozyme?

Answer 20

• Yes,
• RNA is the enzyme that catalyzes the formation of the peptide bones and . . .
• 2/3 of a ribosome is RNA.

Question 21

How do nucleotide sequences in mRNA signel where to start protein synthesis?

Answer 21

• Initiation of protein synthesis requires the presence of several translation initiation factors. The poly A tail must be bound by poly A binding proteins that interacts with eIF4G to ensure that both ends of the mRNA are intact
• 2 GTP hydrolysis steps provide the energy needed.
• A consensus sequence flanking the start codon tells the ribosome to begin translation on this AUG. If the flanking nucleotides differ much , the ribosome will move to the next AUG.This results in alternative starting proints for the protein.

Question 22

How is translation stopped?

Answer 22

• binding of a release factor to an A site containing a stop codon causes termination of translation.
• The peptide is released and in a series of reactions the ribosomal subunits dissociate and are then ready to initate translation again.

Question 23

Why are proteins made on Polyribosomes?

Answer 23

• Upon completion of translation, the ribosomal subunits are near the 5’ end of the mRNA because of the association of the 5’ and 3’ ends of the mRNA. Thus they can restart quickly.

Question 24

How do inhibitors of prokaryotic protein synthesis useful in antibiotics?

Answer 24

• Fungi produce many antibacterial componds that exploit differences in ribosomal subunits.
• Fungi produce all kinds of small molecules that inhibitor bacterial protein synthesis

Question 25

How is the translation of mutated genes with premature stop codons prevented?

Answer 25

* Nonsense mediated decay eliminates mRNAs with premature stop codons. * As mRNA is transported through nuclear pore, a ribosome does a test run on it. * Exon junction complexes (EJC) are bound at exon junctions and are removed by moving ribosomes. * The stop codon should be the last exon, if the ribosome hits a stop codon while EJCs are still present, → activate nonsense mediated decay and the mRNA will be rapidly removed to prevent additional wasted energy.

Question 26

(True/False) Some proteins begin to fold while still being synthesized?

Answer 26

* True

Question 27

* How/When are proteins folded during synthesis?

Answer 27

* As the N terminus emerges from the ribosome, the peptide begins folding almost immediately, aquiring secondary and tertiary structures before the C terminus is complete. * It must be released before final structure can be obtained.

Question 28

What role do Chaperones play in protein folding?

Answer 28

* Many energetially facorable pathways are availble for protein folding. * Chaperones ensure that peptides fold into the correct conformation or to refold them if they get damaged. * The most energetically favorable fold is not always the right (funcational) one.

Question 29

What are hsp70 and how do they work?

Answer 29

* type of chaperone * heat shock proteins recognize hydrophobic stretches of amino acids on the surface of a protein (should be found on the interior) and bind to them. * ATP hydrolysis drives the reaction * They refold misfolded proteins

Question 30

How do hsp60 work?

Answer 30

1. misfolded protein is captured by hydrophobic interactions with opening surface. 2. protein is unfolded 3. misfolded protein is moved into inner space 4. ATP is hydrolyzed and goes through structural modifications to fold correctly 5. ATP binding loosens Cap and allows ejection of the correctly folded protein.

Question 31

What does an exposed section of hydrophoic amino acids indicate? What can they lead to if not corrected?

Answer 31

* Exposed section of hydrophoic amino acids indicates a protein is misfolded * If not corrected the result is usually accumulation of protein aggregates that can be harmful * Dieases of proteopathy include prion disease, Alzheimer's and Parkinson's

Question 32

What does the proteasome do? How is proteasome selective?

Answer 32

* The proteasome searches for misfolded proteins in the nucleus and cytoplasm, ingests them and degrades them so that the AAs can be recycled. * The cap (blue) binds damaged proteins that have been ubuitylated, unfolds them and feeds them into the inner cylinder. * Specialized proteins (E3) recognize misfolded proteins and add multiple ubiquitin peptides to the misfolded peptide.