Topic 11 (Translation) Flashcards
Which four key molecules are involved with translation?
- mRNA
- tRNA
- ribosome
- aminoacyl-tRNA synthetase
What is an aminoacyl-tRNA synthetase?
A set of enzymes that couple each of the 20 amino acids to its appropriate tRNA
How many aminoacyl-tRNA synthetases do we have?
- One per amino acid
How is mRNA read by the ribosome?
In a 3-nucleotide sequence (codon) in the 5’ - 3’ direction
What does degeneracy mean in relation to codons?
More than codon can be used to code for one amino acid
What are the two important parts of a tRNA?
- anticodon loop (3’ - 5’)
- 3’ overhang
Function of the anticodon loop
Matches mRNA sequence to the correct tRNA
Function of the 3’ overhang in tRNA
Amino acid attachment site
True/False? There are 20 different tRNAs
False. Many more (don’t need to know how many)
Prokaryotic ribosome subunits
Small: 30S
Large: 50S
Eukaryotic ribosome subunits
Small: 40S
Large: 60S
True/False? The large ribosomal subunit consists of proteins and rRNA, while the small subunit is only made of proteins
False. Both proteins and rRNA
What is the function of the A site? Full name?
Aminoacyl; binds the incoming tRNA (charged)
What is the function of the P site? Full name?
Peptidyl; holds the tRNA with the growing peptide chain
What is the function of the E site? Full name?
Exit; releases the tRNA formerly bound in the P site
Which amino acid is always synthesized first? Which end (N or C) of the peptide is it located on?
Methionine; N
In the elongation step, which end (N or C) attacks which tRNA/amino acid (located in the A site or P site)?
N of the incoming amino acid (A site) attacks C of the amino acid chain in the P site
How is the ribosome assembled in prokaryotes?
Small subunit + mRNA + f-Met + large subunit (in that order)
ORF
Open reading frame. Stretch of DNA/RNA sequence between a start and stop codon which can be translated into a protein
Polycistronic mRNA
mRNA that contains multiple ORFs
Monocistronic mRNA
mRNA that encodes a single ORF
Prokaryotic mRNA is (polycistronic or monocistronic)
Polycistronic
Eukaryotic mRNA is (polycistronic or monocistronic)
Monocistronic
How many reading frames are possible for each DNA strand?
3
Why are frameshift mutations so disruptive?
A shift in the reading frame may cause a truncated, extended, or a completely different mRNA/protein
What recruits the ribosome to mRNA in eukaryotes?
5’ cap
What is the kozak sequence?
A purine 3 bases upstream of the start codon followed by another G (5’ - [A/G]NNAUGG -3’)
What is the function of the poly-A tail in translation?
It enhances translational efficiency by recruiting translation initiation factors
What are the three common features of tRNA?
- 75-95rnt in length
- 3’ end terminates with CCA (amino acid attachment site)
- sometimes have modified nucleosides
What are the three modified nucleosides that may be used in tRNA? What is a feature of each?
- uridine (uracil + ribose on N1)
- pseudouridine (uracil + ribose on C5)
- dihydrouridine (uracil, but without a double bond between C5=C6)
What are the secondary structure components of tRNA?
- acceptor stem (AA attachment)
- pseudouridine loop
- dihydrouridine loop
- anticodon loop
- variable loop
What parts of tRNA are determinants for tRNA synthetase recognition?
Anticodon loop and acceptor stem
What is the discriminator base?
The most 5’ base on the 3’ overhang on tRNA that distinguishes between amino acids
Describe the steps to tRNA charging by tRNA synthetase
- The carboxyl end of the amino acid attacks the alpha phosphate on ATP
- The adenylated amino acid (bound to tRNA synthetase) is attacked by the 3’OH of the overhang, producing the charged tRNA and AMP
What are the two species of tRNA?
- aminoacyl-tRNA
- peptidyl-tRNA
Aminoacyl-tRNA
Specific amino acid is coupled to its 3’ end. Binds to the A site in the ribosome
Peptidyl-tRNA
Specific amino acid is coupled to its 3’ end along with a chain of amino acids. Binds to the P site in the ribosome
The peptidyl transferase center is contained in:
The large subunit
Function of peptidyl transferase
Peptide bond formation
The decoding center is contained in:
The small subunit
Function of decoding center
Decodes the mRNA codon
A larger S value means what?
Faster sedimentation velocity after centrifugation
What are the peptidyl transferase center and decoding center composed of?
Almost entirely rRNA
True/False? There can only be one ribosome located on each mRNA at a time
False. Multiple may be translating the same mRNA at once
Which end of the polypeptide are new amino acids added onto?
C-term end
Describe the steps to eukaryotic translation initiation
- eIF1, eIF1A, eIF3, and eIF5 bind to 40S (prevent 60S binding and initiator tRNA binding to A site)
- eIF2-GTP brings initiator tRNA to the P site of 40S (43S pre-initiation complex)
- eIF4 factors prepare the mRNA for 43S pre-initiation complex recognition
- The 40S bound to initiator tRNA and mRNA scans for AUG (ATP-dependent step)
- 43S complex releases eIF1, 2-GDP, 3, 4B, and 5 after the initiator tRNA base pairs with the start codon
- binding of eIF5B-GTP to stimulate the association of 60S subunit
- 60S is recruited and initiation factors are released
What is the initiator tRNA?
Met-tRNA
What is the peptidyl-transferase reaction?
Transfer of the growing peptide from peptidyl-tRNA to aminoacyl-tRNA
Describe the steps to eukaryotic translational elongation
- EF-Tu-GTP binds aminoacyl-tRNA at its 3’ end to mask the coupled amino acid from forming a peptide bond prematurely
- correct codon-anticodon interaction occurs in the A site
- EF-Tu-GTP interacts with the factor binding center in 60S
- EF-Tu-GTP undergoes hydrolysis and EF-Tu-GDP + Pi is released
What are the three mechanisms for correct codon recognition?
- Additional H bonds form between 2 A residues of 16S rRNA (small subunit) and the minor groove of the correct codon-anticodon base pairing
- Correct base pairing allows aminoacyl-tRNA bound EF-Tu to interact with the factor-binding center to induce EF-Tu hydrolysis and release
- Correct base pairing allows tRNA to rotate (accommodation) into the correct position for peptide bond formation
What are stop codons recognized by? What happens when they are recognized?
Release factors; hydrolysis of the polypeptide from the peptidyl-tRNA occurs upon recognition
What are class I release factors?
Recognize stop codons and trigger hydrolysis of the polypeptide chain from the tRNA in the P site
How many class I release factors are there in eukaryotes? Which stop codons are recognized by which release factors?
Only one in eukaryotes (eRF1), which recognizes all three stop codons
What are class II release factors?
Stimulate the dissociation of class I release factors from the ribosome after polypeptide chain release
How many class II release factors are there in eukaryotes? Which stop codons are recognized by which release factors?
Only one in eukaryotes (eRF3); all three
Describe the termination step in eukaryotic translation
- RF1 recognizes the stop codon and stimulates polypeptide release through its GGQ motif
- RF3 binds after polypeptide release and displaces the class I RF
- RF3 associates with the factor-binding center of the large subunit to stimulate its own hydrolysis and dissociates from the ribosome
- RRF binds to the A site to recruit EF-G-GTP
- EF-G-GTP stimulates the release of the uncharged tRNAs in P and E sites and may displace the RRF from the A site
- EF-G-GDP and mRNA are released from the ribosome
Which steps of translation require ATP hydrolysis? GTP hydrolysis?
tRNA charging: ATP
eIF2 (initiation): GTP
mRNA scanning for start codon: ATP
eIF5B (recruits large subunit): GTP
EF-Tu (elongation): GTP
RF3 (termination): GTP
EF-G (termination): GTP
