Topic 11 Flashcards
The 3 key components of translation are mRNA (information carrying molecule), tRNA (amino acid adaptor molecule) and the ribosome (amino acid linking complex). What is the fourth major translational component?
Aminoacyl-tRNA synthetases: a set of enzymes that couple each of the 20 amino acids to its appropriate tRNAs (charge the tRNAs)
What is the genetic code?
Information stored in mRNA is read as a triplet code, and each triplet code consists of a 3 nucleotide codon known as the genetic code.
True or false: the genetic code is degenerate
True
- The third nucleotide can wobble, allowing for more than one codon to code for a particular amino acid
Describe the structure of tRNA in general, and what 2 important parts make up the molecule
Single stranded RNA folded into cloverleaf structure by intramolecular base pairing
2 important parts:
- Anticodon loop recognizes and base pairs with the mRNA
- 3’ amino acid attachment site
Prokaryote small and large subunits of ribosome
Small subunit: 30S
Large subunit: 50S
Eukaryote small and large subunits of ribosome
Small subunit: 40S
Large subunit: 60S
Ribosomes are composed of…
One small and one large subunit, that each have: RNAs and proteins
Large subunit of ribosome function
Contains the peptidyl transferase center for peptide bond formation
Small subunit of ribosome function
Contains the decoding centre for decoding the mRNA codon
What are the three sites of the ribosome and describe them
- A (aminoacyl/acceptor) site
- incoming tRNA bound - P (peptidyl) site:
- growing polypeptide chain - E (exit) site:
- releasing tRNA
Define the ORF
A stretch of DNA or RNA sequence between a start codon and a stop codon which can be translated into a protein
Eukaryotic start codon
AUG (methionine/Met/M)
Eukaryotic stop codons
UAA, UGA, UAG
Polycistronic mRNAs
mRNAs containing multiple ORFs (i.e. single mRNA codes for multiple proteins)
- e.g. prokaryotic mRNAs
Monocistronic mRNAs
mRNAs encoding a single ORF
- e.g. eukaryotic mRNAs
How many possible reading frames are there for each DNA strand?
3
What is required on eukaryotic mRNA for recruiting ribosomes?
The 5’ cap
What does the ribosome do first after binding to the mRNA?
It scans from 5’ to 3’ until it encounters the start codon
Kozak sequence
Purine (A or G) 3 bases upstream of the start codon (AUG) followed by another ‘G’ (5’-G/ANNAUGG-3’) which increases translational efficiency
True or false: translation cannot take place without the Kozak sequence in eukaryotes
False
- Translation can take place without the Kozak sequence but it won’t be as efficient
What does the poly-A tail at the 3’ end of mRNA ensure, other than preventing 3’ to 5’ degradation?
Enhances the translational efficiency by recruiting key translation initiation factors
What are the 3 common features of tRNA?
- 75-95 ribonucleotides in length
- 3’ end terminates with CCA sequence, serving as the site for amino acid attachment
- A subset of tRNAs have modified nucleosides in their primary structure
What are three examples of modified nucleosides in tRNA primary structure?
- Uridine
- Pseudouridine (ΨU)
- Dihydrouridine (D)
Have to do with presence/placement of double bond in base
Draw out a tRNA molecule in secondary structure and label the following regions:
- Acceptor stem
- ΨU loop
- D loop
- anticodon loop
- variable loop
Acceptor stem on tRNA
Site for amino acid attachment
ΨU loop on tRNA
Has pseudouridines in the loop
D loop on tRNA
Has dihydrouridines in the loop
Anticodon loop
Contains 3 nucleotide sequence responsible for recognizing the codon by base pairing with the mRNA
Variable loop
Variable in size
What are the two determinants for tRNA synthetase recognition on a tRNA molecule?
Acceptor stem and anticodon loop
Aminoacyl-tRNA synthetases attach an amino acid to a tRNA in 2 enzymatic steps:
- Give the steps for tRNA charging
Adenylation of amino acid and tRNA charging
1. Carboxyl end of amino acid attacks alpha phosphate of ATP which adenylates the amino acid
2. 3’OH of tRNA attacks carbon on carboxyl group of adenylated amino acid which is bound to the tRNA synthetase, releasing charged tRNA and AMP
True or false: only one tRNA synthetase attaches each amino acid to all of the appropriate tRNAs
True
- There are 20 different tRNA synthetases, one for each amino acid!
Difference between aminoacyl-tRNA and peptidyl-tRNA?
Both have a specific amino acid coupled to its 3’ end, but aminoacyl-tRNA is bound to the A site while peptidyl-tRNA is bound to the P site
Svedberg Unit
Larger the S value = faster the sedimentation velocity and larger the mole
- Larger S means that it settles down to bottom of test tube faster after centrifugation
Sedimentation velocity is determined by what two things?
Both shape and size
What are the two core functional domains of the ribosome and what are they composed almost entirely of?
The peptidyl transferase centre and the decoding centre, which are composed almost entirely of rRNA
True or false: Svedberg units are additive
False
- e.g. 60S + 40S is not equal to 100S ribosome in eukaryotes. 80S ribosome is present in eukaryotes
Large and small subunits undergo ______ during each translational cycle
Undergo association and dissociation during each translational cycle
True or false: Each mRNA can be translated simultaneously by multiple ribosomes
True
New amino acids are attached to which terminal of the growing polypeptide chain?
C-terminus
What are the 3 general steps of eukaryotic translational initiation?
- Binding of the initiator tRNA (met-tRNA) to 40S before association with the mRNA
- Auxiliary factors mediate mRNA recognition (helps recruitment of the large subunit and makes the AUG exposed), and 40S scans for start codon
- 60S is recruited after the initiator RNA base pairs with the start codon
What is recruited to the ribosome during eukaryotic translation initiation to prevent 60S binding and tRNA binding to the A site?
eIF1, eIF1A, eIF3, and eIF5
Function of eIF2-GTP in eukaryotic translation initiation?
eIF2-GTP brings the tRNA(Met) to the P site of 40S to form 43S pre-initiation complex
Function of eIF4 factors in eukaryotic translation initiation?
- Which eIF4 factors specifically?
eIF4 factors prepare the mRNA for the 43S pre-initiation complex recognition
- eIF4G, eIF4A, eIF4B
What does the pre-initiation complex do to look for the first start codon on the mRNA?
- The pre-initiation complex moves along the mRNA in a 5’ to 3’ direction in an ATP-dependent manner to look for the first start codon
What occurs to the 43S pre-initiation complex once the start codon is detected?
43S complex undergoes conformational changes to release eIF1, eIF2-GDP, eIF3, eIF4B and eIF5
Binding of what molecule stimulates the association of the 60S subunit?
Binding of eIF5B-GTP
What does binding of the 60S subunit lead to further release of?
Binding of the 60S subunit leads to further release of the initiation factors
tRNA Met sits in which site of the ribosome?
The P site
What are the 3 elongation steps in translation?
- Loading of aminoacyl-tRNA to the A site
- Formation of peptide bond between aminoacyl and peptidyl tRNA (peptidyl transferase reaction: transfer of the growing polypeptide from peptidyl-tRNA to aminoacyl-tRNA)
- Translocation of aminoacyl-tRNA to the P site
Role of EF-Tu in translation elongation
Aminoacyl-tRNA is bound to EF-Tu-GTP at its 3’ end to mask the coupled amino acid from forming a peptide bond (helps position amionacyl-tRNA in A site). Basically, its role is to stabilize the aminoacyl-tRNA by preventing the amino acid at the 3’ end from prematurely reactng and forming a peptide bond before proper alignment in the ribosome.
- When the correct codon-anticodon interaction occurs, EF-Tu interacts with the factor binding centre in the 60S subunit and undergoes hydrolysis
- EF-Tu-GDP is released
What are 3 mechanisms for correct codon recognition?
- Additional hydrogen bonds formed between adenine residues of the 16S rRNA (prokaryotic version of rRNA) and the minor groove of the correct anticodon in the tRNA
- 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 (accomodation) into the correct position for peptide bond formation
When stop codons enter the A site, the codons are recognized by release factors that activate the hydrolysis of the polypeptide from the peptidyl-tRNA. What are the 2 classes of release factors (RFs)?
Class I: recognizes the stop codons and triggers the hydrolysis of the peptide chain from the tRNA in the P-site
- There is only 1 class I RF (eRF1) in eukaryotes to recognize all the 3 stop codons
Class II: Stimulates the dissociation of Class I from the ribosome after polypeptide chain release
- There is only 1 class II Rf (eRF3) in eukaryotes to recognize all the 3 stop codons
Describe the steps of RF binding to the A site during translation termination (5 steps)
- RF1 (Class I RF) recognizes and binds to the stop codon
- RF1 stimulates the polypeptide release through its GGQ motif
- RF3 (Class II RF) 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
- RF3-GDP dissociates from the ribosome
Describe the steps of translation termination after RF3-GDP dissociates from the ribosome
- Deacylated tRNAs still bound to the P and E sites
- RRF (Ribosome Recycling Factor) binds to the A-site to recruit EF-G-GTP
- EF-G-GTP stimulates the release of uncharged tRNAs in the P and E sites, and may displace the RRF from the A-site
- EF-G-GDP and mRNA are released
