Translation Flashcards
Briefly state what translation is
translating the language of RNA nucleotides into the language of amino acids
What is translated?
mRNA
What is mRNA translated into?
a linear chain of amino acids
Where does translation occur?
in the cytoplasm
What makes the reactions in translation occur?
the catalytic activity of ribosomes (aka ribozymes)
What else can ribosomes be called? why?
ribozymes because they contain RNA which has catalytic activity
What is a codon? Give an example
a triplet of nucleotides in the mRNA
ex. AUG
How many amino acids does each codon code for?
one amino acid
T or F: codons can overlap
False, they are non-overlapping
T or F: the genetic code is nearly universal
True
How many codon possibilities are there?
64
Why is the genetic code redundant?
there are 64 possible codons but only 20 amino acids, so some amino acids are coded by multiple codons
What is the start codon? What does it code for?
AUG is always the first codon read from the mRNA transcript
it codes for methionine
How many start codons are there?
1
How many stop codons are there?
3
What are the stop codons? what do they code for?
UAA, UAG, UGA
they signify the end of the coding region of the mRNA and the end of the polypeptide
they DO NOT CODE FOR ANY AMINO ACID
T or F: the stop codons code for specific amino acids
FALSE
they just mean it’s the end of the mRNA transcript, no amino acid is added
Why is it critical that codons are read in the correct frame? how is the correct frame ensured?
ensured by the initiation process orienting the AUG codon in a specific position in the ribosome
crucial because if translation begins even one letter after, a completely different sequence will be translated
ex. The red dog ate the big cat –> her edd oga tet heb igc at if you start one letter later
What is main function of tRNA?
it is the molecule that translates the mRNA sequence into amino acid sequence
Describe the structure of tRNA
an RNA molecule with extensive intrachain binding which results in a cloverleaf-like structure that twists into a 3D upside down ‘L’
T or F: most tRNAs are different lengths
false, they are all about the same length (73-93 nucleotides)
Why do tRNA molecules have many unusual bases?
post-transcriptional changes like methylation
How is the ‘stem and loop’ structure of a tRNA molecule formed?
the base sequences in one part of the molecule are complementary to a nearby sequence
What is an anticodon?
a 3-letter sequence that tRNA has that is complementary and antiparallel to a codon in the mRNA
T or F: tRNA contains specific anticodons that are only antiparallel to the mRNA codon
FALSE! they are antiparallel AND complimentary to the codon
How is aminoacyl-tRNA made?
each tRNA is covalently linked to a specific amino acid at the tRNA’s 3’ end
What is another term for the 3’ end of a tRNA?
acceptor stem
What is covalently linked to the 3’ end of a tRNA? What does this form?
a specific amino acid
this forms an aminoacyl-tRNA
How are the correct polypeptide sequences made?
interactions between successive codons in the mRNA and the anticodons of the aminoacyl-tRNA
What amino acid will be joined to the tRNA with the anticodon:
5’ - AAG - 3’
3’ - UUC - 5’
Read: 5’ - CUU - 3’
CUU codes for leucine
T or F: the codon will be read in the direction it is given, even if antiparallel 3’-5’
false! it will always be read 5’-3’
What amino acid will be joined to the tRNA with the anticodon:
3’ - CAU - 5’
5’ - GUA - 3’
GUA codes for Val
Describe the Wobble Hypothesis
some tRNA molecules require accurate base pairings at only 2 of their 3 codon positions (the first 2) and they can tolerate a mismatch on the 3’ position
the 3’ position is called the wobble position
ex. if the wobble base is a U, it can bind to either an A or a G
What does the Wobble Hypothesis explain?
why some amino acids can be coded for by multiple codons
why the position in the codon that varies is the last position (looking at the genetic code table)
ex. CUU CUC CUA CUG all code for Leu and it's only the third letter that's different - the wobble position
What is the purpose of the wobble hypothesis?
it limits the number of distinct tRNAs needed to bind to the 64 codons
there are 64 codons, but only 32 tRNA molecules
ex. 4 leucine codons require only 2 distinct tRNA molecules
Is the wobble in the codon position or the anticodon?
CODON
What are the 4 main steps of translation?
- correct amino acids attached to tRNA molecules by aminoacyl-tRNA Synthetase
- initiation of protein synthesis
- elongation
- termination of peptide synthesis
What is aminoacyl-tRNA Synthetase? explain its function
an enzyme that interacts with both the anticodon region and the 3’ acceptor stem
it ensures the correct amino acids are attached to the tRNA with a given anticodon
what is a charged tRNA?
a tRNA molecule + an amino acid
What is the first step in attaching an amino acid to the tRNA?
an amino acid is attached to an ATP molecule and together they bind to an aminoacyl-tRNA Synthetase
What happens after the amino acid + ATP bind to aminoacyl-tRNA Synthetase?
2 inorganic phosphates (pyrophosphate) are released
amino acid + AMP are bound to the active site on aminoacyl-tRNA synthetase
Where are the amino acid + AMP transferred to after the ATP is cleaved?
from the aminoacyl-tRNA synthetase to the tRNA
What happens to the AMP after it and the amino acid are transferred to the tRNA?
AMP dissociates
What happens to the amino acid + tRNA + aminoacyl-tRNA synthetase once the AMP leaves?
the tRNA + amino acid dissociate from aminoacyl-tRNA synthetase
tRNA is now charged with an amino acid
What happens to most of the energy released from ATP during the process of attaching an amino acid to tRNA?
it is preserved in the aminoacyl-tRNA covalent bond
What are the 3 ribosome sites in translation initiation? Where are they located?
Aminoacyl tRNA site
Peptidyl tRNA site
Exit site
Both the large and small ribosomal subunits contribute to the A and P sites
the E site is mostly in the large ribosomal subunit
What is unique about translation initiation in prokaryotes?
it involves a specific mRNA sequence called the Shine-Dalgarno sequence
Describe how translation is initiated in prokaryotes
complementary rRNA in the small ribosomal subunit binds to the Shine-Dalgarno (mRNA) sequence to line the P site directly over top of the AUG codon
Describe the process of translation initiation in eukaryotes
the ribosome binds with initiation factors at the 5’ cap and scans until it finds the first AUG codon
T or F: both prokaryotes and eukaryotes have a Shine-Dalgarno sequence
False, only prokaryotes
What is the Shine-Dalgarno sequence?
A specific mRNA sequence only present in prokaryotic translation initiation
What makes the initation AUG codon different from other AUG codons?
it has a foryml group in the middle of the protein
How many methionine tRNAs are there? why?
2
one for the formylmethionine (fmet)
one for the other methionines
What is fmet? Where is it located?
the methionine tRNA for the formylmethionine (initiation methionine)
located at the amino terminus
What does a formyl group look like?
a H-C=O attached by the C to the N of the amino acid
Where is the other methionine tRNA located?
in the interior of the polypeptide
What 5 things does translation initiation require in bacterial cells?
30S and 50S ribosomal subunits
the mRNA transcript
tRNA bound to fMet
three initiation factors
GTP as an energy source
What are the 3 initiation factors for bacterial translation initiation?
IF1
IF3
IF2-GTP
What ribosomal subunits are required for bacterial translation?
30S small
50S large
What is IF1 and what is its function?
A translation initiation factor
blocks the A site to prevent new tRNAs from binding
What is IF3 and what is its function?
a translation initiation factor
binds to the E site to prevent large ribosomal subunit from binding
What is IF2-GTP and what is its function?
a translation initiation factor that binds to the P-site and recruits the initiator tRNA (tRNA-fMet)
What is the function of the Shine-Dalgarno sequence?
this sequence of mRNA transcript aligns with the 16S rRNA in the small subunit to position the AUG codon over the P Site
What happens when the 50S large subunit associates (bacterial translation initiation)?
GTP is cleaved
What does the cleaving of GTP cause (after the 50S subunit binds)? what does it form?
all the Initiation factors dissociate and the E and A sites are open
the 50S subunit can now come on
this forms the initiation complex
What is included in the initiation complex?
the large 50S subunit an open E and A site A tRNA-fMet bound to the P site the Shine-Dalgarno sequence above the AUG initiator codon the 30S subunit holding it in place
What is included in the initiation complex?
the large 50S subunit
an open E and A site
A tRNA-fMet bound to the P site
the Shine-Dalgarno sequence above the AUG initiator codon
the 30S subunit
What 4 things does elongation in bacterial cells require?
the initiation complex with tRNA-fMet in the P site
free floating aminoacyl-tRNAs (charge tRNAs)
3 soluble cytosolic elongation factors
GTP
What are the 3 soluble cytosolic elongation factors?
EF-Tu
EF-Ts
EF-G
What are the 3 substeps of elongation?
binding of incoming aminoacyl-tRNA in the A site
peptide bond formation
translocation of ribosome
How does incoming aminoacyl-tRNA bind to the A site?
aminoacyl-tRNA binds to GTP-bound EF-Tu
aminoacyl-tRNA + GTP + EF-Tu enter the A site with the correct anticodon
GTP is cleaved –> GDP + EF-Tu leave
Aminoacyl-tRNA is now in the A site
Where does the peptide bond form during elongation?
between the 2 amino acids in the A and P sites
remember that the P site has the tRNA-fMet and the A site now has an aminoacyl-tRNA
How does the peptide bond form during elongation? What is the result?
the fMet on the tRNA in the P site transfers to the amino group of the aminoacyl-tRNA in the A site
result = tRNA in the A site bound to a dipeptide and an uncharged tRNA in the P site
What catalyzes the peptide bond formation?
the 23S rRNA in the large subunit
What is the purpose of the peptide bond formation during elongation?
transferring the fMet from the tRNA in the P site to the tRNA in the A site creates a growing chain of amino acids (a dipeptide)
What happens after the peptide bond is formed?
TRANSLOCATION:
the ribosome moves one codon towards the 3’ end of the mRNA
Describe translocation in elongation
after the peptide bond forms, the ribosome moves one codon toward the 3’ end of the mRNA
How does translocation of the ribosome effect the tRNA molecules in the EPA sites?
the (empty) tRNA from the P site moves to the E site
the tRNA (with the dipeptide) from the A site moves to the P site
the A site is open and ready for a new tRNA
What happens to the tRNA that is moved to the E site after translocation?
it is empty and it is released into the cytosol
What elongation factor does translocation require?
EF-G translocase bound to GTP
How does EF-G translocase function in translocation?
it is bound to GTP
it hydrolyzes the GTP and induces a conformational change
this provides the energy to move the ribosome downstream one codon
What happens to the EF-G translocase after GTP hydrolysis?
it is now EF-G-GDP and it leaves through the open A site
What happens when the A site is empty? (after translocation)
the elongation cycle:
binding new charged tRNA in A site
peptide bond formation
ribosome translocation
repeats over and over again
During elongation, how many GTPs are hydrolyzed for each amino acid correctly added to the growing polypeptide?
two GTPs hydrolyzed to GDP + Pi as the ribosome moves from codon to codon along the mRNA
T or F: translation elongation does not require much energy
False!
2 GTP molecules are required per amino acid added
ex. if you have an 800 amino acid long chain, you’d need 1600 GTPs to build that protein
Where does the polypeptide chain exit the ribosome?
through a tunnel in the large ribosomal subunit
How long will elongation continue?
until termination is signalled by the stop codons (UAA, UAG, UGA)
What happens when a stop codon is reached?
the stop codons bind to a release factor protein called RF
What happens when the stop codon binds to the RF?
the RF hydrolyzes the tRNA-peptidyl bond between the last amino acid and the tRNA in the P site to release the complete peptide
T or F: the release factor mimics a tRNA
true
What does hydrolyzing the tRNA-peptidyl bond cause?
the dissociation of the tRNA-peptidyl bond between the last amino acid and the tRNA in the P site
the release of the final tRNA
How is the RF dissociated from the mRNA? What else does this dissociate?
GTP hydrolysis dissociates the RF from the mRNA
AND dissociates the 2 ribosomal subunits
Describe polyribosomes
a single mRNA molecule is being translated by multiple ribosomes simultaneously
Can polyribosomes be seen under microscopes?
yes
T or F: polyribosomes only occur in prokaryotes
false, they occur in both prokaryotes and eukaryotes
Explain why prokaryotic and eukaryotic polyribosomes look different
Prokaryote: transcription and translation occur at the same time so as RNAP is synthesizing RNA, the ribosomes are also translating it
eukaryote: the transcript is made first and then many ribosomes are working to translate it
T or F: almost all proteins are synthesized on ribosomes that are initially in the cytosol
true
T or F: proteins all use the same mechanisms for travelling to their final cellular destinations
False, they all have different mechanisms
What do all proteins have that allow them to travel to their cellular destinations?
a signal sequence within their primary structure
What are 6 examples of final cellular destinations for proteins synthesized on ribosomes in the cytosol?
secretion/integration in plasma membrane
lysosome
mitochondria
chloroplast
nucleus
peroxisome
What type of signal sequence imports proteins into the ER?
N-terminal sequence
What type of signal sequence returns proteins back to the ER?
C-terminal sequence
The presence of a ____ signal peptide at the ____ of the protein targets ribosomes to the ____ ER membrane
presence of an N-TERMINAL signal peptide at the BEGINNING of the protein targets ribosomes to the ROUGH ER membrane
Describe co-translational transport
transportation of a protein that occurs during translation
What kind of proteins would be co-translationally transported?
those with an N-terminal signal peptide at the beginning of the protein
What happens to the remainder of a protein when it begin with the N-terminal signal peptide and it is transported to the rough ER?
the remainder of the protein will be made and leave the ribosome directly into the ER lumen
What happens to proteins that do not have an N-terminal signal peptide?
they can still be trafficked when translation is complete
ex. they may have an NLS and be transported to the nucleus
Describe post-translational transport
transportation of a protein after translation is complete (the protein is fully sequenced, translated, and folded)
What 4 types of proteins have an ER signal sequence?
any protein requiring post-translational modifications
integral membrane proteins
secretory proteins
resident proteins of the ER, Golgi, lysosomes or other Endomembrane system component
What happens as soon as a signal peptide is translated?
it is recognized by a signal recognition particle (SRP)
What happens after the SRP recognizes the signal peptide?
remember this occurs DURING translation:
the SRP brings the entire ribosome (+ the growing polypeptide attached to the signal peptide) to an SRP receptor on the ER membrane and the SRP binds to the receptor
What does the SRP binding to the SRP receptor do?
positions the ribosome to dock at an adjacent translocon channel
What is an SRP?
Signal Recognition Particle
it recognizes the signal peptide for the ER and brings the ribosome to the ER membrane
How is the remainder of a protein synthesized once the SRP has bonded to the SRP receptor on the ER membrane? What is the purpose of this?
through a pore in the translocon
so the polypeptide is translocated across the ER membrane into the ER lumen
What is a translocon?
A channel on the ER membrane that allows a protein from a cytosolic ribosome to enter the lumen of the ER while it is being translated (Co-translation translocation)
What are both the SRP and SRP receptors bound to?
GTP
How does the SRP dissociate from the SRP receptor?
both GTPs bound to the SRP and SRP receptor hydrolyze and the SRP dissociates
What membrane protein is associated with the translocon?
signal peptidase
What is the function of signal peptidase?
it cleaves the signal sequence of the growing polypeptide as it passes through the translocon and the sequence is degraded
T or F: translation of the polypeptide continues through the translocon into the ER lumen
true
What happens to the translocon after translation is complete?
it closes
What happens to the protein in the ER lumen after translation is complete?
it folds into its final conformation
Where do most proteins eventually move to? what do they do this through?
eventually move to the Golgi lumen via vesicles
What does a protein that is supposed to stay in the ER contain?
a retention sequence: K-D-E-L at the C-terminal end
What is the retention sequence? where is it located?
K-D-E-L
at the C-terminal end
What happens to proteins with KDEL that accidentally get shipped forward to the Golgi?
they get returned to the ER
How does the process differ for proteins destined for membranes?
the translocon will close before translation of the polypeptide is complete so that it will slide out into the membrane and embed there
the stop transfer sequence will be embedded in the membrane and the remainder of the protein will be synthesized in the cytosol
Where will a secretory protein will end up?
completely inside the ER lumen and will eventually be expelled from the outside of the cell
What are the different ‘parts’ of an integral membrane protein?
early part will be in the ER lumen
at least one part will be embedded in the membrane
one part will be cytosolic
If part of a protein that is destined to be an integral membrane protein is in the ER lumen, where will it be when it reaches the plasma membrane?
in the extracellular space
If part of a protein that is destined to be an integral membrane protein is in the cytosol, where will it be when it reaches the plasma membrane?
in the cytosol
Which terminal end is cytosolic in membrane proteins? without modifications
N terminal
Which terminal end is in the lumen in membrane proteins? without modifications
C terminal
