Translation Flashcards
1
Q
how does eukaryotic protein synthesis primarily differ from prokaryote protein synthesis?
A
Eukaryotic protein synthesis differs from bacterial protein synthesis primarily in translation initiation
2
Q
how is prokaryote transcription and translation related to each other?
A
Transcription and translation are closely coupled
The primary transcript serves as mRNA and is used immediately as the template for protein synthesis
3
Q
ribosome
A
the site of protein synthesis
4
Q
what is the structure of the ribosome (subunits and centrifugation)?
A
The E. Coli ribosome sediments under centrifugation at 70S (sediment coefficient) and can be dissociated into 2 subunits…
A large 50S subunit and a smaller 30S subunit
5
Q
what is the ribosomal large 50S subunit composed of?
A
34 proteins
23 rRNA
5S rRNA
6
Q
what is the ribosomal small 30S subunit composed of?
A
21 proteins
16S rRNA
7
Q
what are the 3 different types of E. Coli ribosomal RNA (rRNA)?
A
5S rRNA
16S rRNA
23S rRNA
8
Q
what role do the rRNAs play in protein synthesis and ribosomal structure?
A
2/3 of the mass of ribosomes is RNA → critical for the structure and function of the ribosome
The rRNAs fold into complex structures that have many short duplex regions
rRNA is the ACTUAL catalysts for protein synthesis; ribosomal proteins make only a MINOR contribution
9
Q
what are the 3 steps of protein synthesis?
A
- Initiation
- Elongation
- Termination
10
Q
how many tRNA binding sites do each ribosome have and what do they do?
A
3 tRNA binding sites: A, P, E site
At each site, the tRNA is in contact with BOTH the 30S subunit (which holds the mRNA template) and the 50S subunit (which catalyzes the formation of peptide bonds)
11
Q
What does the A site stand for and its function?
A
Aminoacyl site
- binds to the incoming aminoacyl-tRNA which carries a single amino acid that corresponds to the codon on the mRNA
12
Q
What does the P site stand for and its function?
A
Peptidyl site
- holds the tRNA with the growing polypeptide chain; this is where the peptide bond formation occurs and the chain is extended with each addition
13
Q
What does the E site stand for and its function?
A
Exit site
- after the tRNA transfers its peptide chain, it moves to the E site where it is uncharged (aka no amino acid is attached) and now ready to exit the ribosome –> this frees up space for the next tRNA to enter the A site and continue the cycle again
14
Q
what is the polypeptide exit tunnel?
A
The peptidyl transferase center in the 50S subunit facilitates the formation of a peptide bond between the amino acid in the A site and the growing peptide in the P site –> this reaction elongates the polypeptide chain, which begins to fold as it exits the ribosome
–> Polypeptide Exit Tunnel: After bond formation, the growing polypeptide chain passes through a tunnel in the 50S subunit, eventually reaching the back of the ribosome
- This tunnel guides the nascent chain as it emerges/exits from the ribosome (protects it from the cellular environment and allows initial folding to begin within the ribosome)
15
Q
how does each tRNA molecule contact the 30S and 50S subunits?
A
Each tRNA molecules contacts BOTH the 30S and 50S subunit
16
Q
the tRNA molecules in which sites are base paired with the mRNA?
A
the tRNA molecules in sites A and P are base paired with mRNA
17
Q
What is the difference between the 50S and 30S subunits functions and structure?
A
50S:
function: primarily responsible for catalyzing peptide bond formation –> (contains the PEPTIDYL TRANSFERASE CENTER) where amino acids are joined together to form a polypeptide chain
- contains an exit tunnel through which the newly formed polypeptide chain exits the ribosome
tRNA binding: binds the acceptor end of the tRNA in both the A and P sites and positioning them near the peptidyl transferase center to allow for peptide bond formation between the amino acids
structure: more rigid structure such to provide a stable environment for peptide bond formation
30S:
- primarily involved in decoding the mRNA sequence by binding to the mRNA template and aligning the mRNA codons with the correct tRNA anticodons to ensure the correct sequence of amino acids –> performs this function by interacting with the anticodon loops of the tRNA at each tRNA binding site (A,P,E) and verifying correct base pairing
tRNA binding: interacts with the anticodon region of the tRNA at the A, P, E sites and aligns it with the codon on the mRNA
structure: more flexibly structure and allows it to adjust and accommodate the mRNA and tRNA movements during translation
18
Q
what are considered the ACTUAL catalysts for protein synthesis?
A
rRNA (ribosomal proteins only make a minor contribution)
19
Q
what is an aminoacyl-tRNA
A
a tRNA with an amino acid attached
20
Q
where does the polypeptide chain exit the ribosome?
A
This polypeptide chain exits through a tunnel in the 50S subunit. The channel is positioned at the back of the ribosome, emerging from the 50S subunit at the end of the P site.
The exit channel is a narrow pathway that allows the newly synthesized protein to begin folding as it emerges, preparing it for further structural formation once translation is complete. This channel is essential in guiding the polypeptide chain while preventing it from misfolding or interacting prematurely with cellular components.
21
Q
what is the usual start signal for translation in bacteria?
A
The start signal is usually AUG preceded by several bases that pair with 16S rRNA
22
Q
polycistronic definition
A
a single mRNA can contain multiple coding regions where each region encodes a different protein (thus aka a single mRNA can encode for MULTIPLE proteins)
→ each of the coding regions has its own initiation site (including its own Shine-Dalgarno sequence and start codon) which allows the ribosome to initiate translation at multiple points along the mRNA
- many mRNAs in bacteria are polycistronic
23
Q
what is the first codon usually to be translated and what amino acid does it code for?
A
codon AUG –> amino acid methionine
24
Q
Initiation in bacteria begins at least how many nucleotides downstream of which end of the mRNA?
A
Initiation in bacteria begins at least 25 nucleotides downstream of the 5’ end of the mRNA
25
5' untranslated region (5' UTR) and what sequence does it contain?
the segment of mRNA/nucleotides that lies between the 5’ end of the mRNA and the start codon (aka the first codon translated)
- this region is not translated into protein but contains REGULATORY ELEMENTS essential for the initiation of translation
- contains the Shine-Dalgarno sequence
26
shine-dalgarno sequence and function
located within the 5' UTR and is a purine-rich sequence approximately 10 base pairs upstream of the start site that interacts with the 16S rRNA to correctly position the AUG codon in the A site (and directs the protein synthesis machinery to the start site)
- the Shine-Dalgarno sequence pairs with the complementary sequence on the 16S rRNA --> pairing serves as an anchor for the ribosome and allows it to locate the nearby start codon and ensures correct alignment to initiate translation
- aka helps recruit the ribosome to the mRNA by interacting with the 16S rRNA and facilitating the correct alignment of the start codon with the ribosome's A site
27
what are the 2 kinds of interactions that determine where protein synthesis starts?
1. The pairing between the mRNA bases and the 3’ end of 16S rRNA (aka the Shine-dalgarno and 16S rRNA interaction which anchors the ribosome near the start codon)
2. The pairing between the initiator codon on the mRNA and the anticodon on the initiator tRNA (the initiator codon and initiator tRNA pairing which precisely positions the first amino acid of the protein)
28
How does the pairing between the mRNA bases and the 3’ end of 16S rRNA help determine where protein synthesis starts?
Shine-Dalgarno Sequence (a short, purine-rich region in the 5' untranslated region (5’ UTR) of bacterial mRNA, typically located about 10 nucleotides upstream of the start codon AUG) is complementary to a region at the 3' end of the 16S rRNA in the 30S ribosomal subunit
base pairing with 16S rRNA:
The ribosome binds (base pairs) to the mRNA, and the Shine-Dalgarno sequence pairs with the complementary sequence on the 16S rRNA
--> this pairing serves as an "anchor" for the ribosome, allowing it to locate the nearby start codon and ensuring that it aligns the mRNA correctly for the initiation of translation
Positioning the Start Codon in the A Site: The Shine-Dalgarno/16S rRNA pairing positions the start codon in the ribosome's P site --> setting up the first codon for decoding
- Without this interaction, the ribosome might not bind precisely, leading to translation errors or failure to initiate
29
how does the pairing between the initiator codon on the mRNA and the anticodon on the initiator tRNA help determine where protein synthesis starts?
The initiator codon AUG is recognized specifically by an initiator tRNA that carries the first amino acid of the nascent polypeptide chain
--> The initiator tRNA, loaded with formyl-methionine in bacteria, has a specialized anticodon loop that pairs exclusively with the AUG start codon on the mRNA
- Unlike other tRNAs, the initiator tRNA is designed to bind DIRECTLY to the ribosome's P site (rather than entering through the A site as with elongation tRNAs)
- The formyl group on methionine (in bacteria) distinguishes it as the initiating amino acid, marking the beginning of the polypeptide chain
The pairing of the initiator codon with the anticodon of the initiator tRNA finalizes the positioning for translation to begin and with the help of initiation factors, the large ribosomal subunit 50S joins the complex and forms the complete ribosome --> now ready to elongate the nascent polypeptide chain
30
what is N-formylmethionine (fMet) and how is it activated?
N-formylmethionine (fMet): a modified form of methionine by the addition of a formyl group CHO- to its amino group and is the initiator amino acid in most proteins in bacteria
- the formyl group is added to the methionine after it is attached to the tRNA and serves as a marker for the starting amino acid --> helps distinguish it from other methionines added later into the protein sequence
- the formyl group si typically removed after the protein is synthesized
fMet is activated by attachment of the initiator tRNA
31
initiator tRNA
a specialized tRNA molecule that delivers the FIRST amino acid of the protein (typically methionine; or formyl-methionine in bacteria) to the ribosome at the start of translation
Structure: it is structurally unique compared to elongator tRNAs which ADD amino acids during elongation
--> the unique structure allows it to bind DIRECTLY to the P SITE instead of the A site (which is the entry point for all other tRNAs)
32
fMet-tRNA function
binds ONLY to the initiation codon (AUG) and NOT to AUG codons elsewhere in the mRNA
33
tRNAm function
recognizes the internal codons for methionine (not the start AUG codon)
34
what is the difference and similarities between tRNAf and tRNAm?
similarities:
- the same synthetase (enzyme methionyl-tRNA synthetase) charges BOTH tRNAm and fMet-tRNA with methionine
--> this aminoacyl-tRNA synthetase recognizes methionine and attaches it to both the initiator and elongator tRNAs
BUT then after methionine is attached to fMet-tRNA by methionyl-tRNA synthetase, a 2nd enzyme called transformylase specifically recognizes tRNAᶠᴹᵉᵗ and catalyzes the addition of a formyl group to the methionine, creating formylmethionine (fMet) --> this modification enables tRNAᶠᴹᵉᵗ to participate exclusively in the initiation of translation
In bacterial translation, there are 2 types of tRNA molecules that carry the amino acid methionine: fMet-tRNA and tRNAm
- These tRNAs are specialized to ensure that the methionine at the start of a protein (the initiator methionine) is recognized separately from methionines incorporated later in the sequence
fMet-tRNA
- the initiator tRNA that specifically and ONLY binds to the START codon (AUG) at the beginning of an mRNA sequence to initiate translation in bacteria and ensures that methionine is added specifically at the INITIATION SITE (P site) and not within the protein sequence
- in bacteria, the methionine attached to fMet-tRNA is modified by the addition of a formyl group -->formylmethionine (fMet)
- This modification signals to the ribosome that this methionine is the initiator and should be incorporated only at the start of the protein (the formyl group is typically removed from the protein after translation begins, meaning it doesn't remain in the final protein structure)
tRNAm
- a tRNA that also carries methionine but is designed to recognize INTERAL AUG codons within the mRNA sequence and NOT the start codon --> helps ensure that methionine can be added at various points within a protein
- tRNAm enters the ribosome's A site during elongation (not the P site like fMet-tRNA)
- tRNAm carries a regular methionine without a formyl group
35
Initiation factors (IF) in bacteria function
assist in the assembly of the protein-synthesizing initiation complex (30S and then 70S)
36
Bacterial protein synthesis is initiated by ___
Bacterial protein synthesis is initiated by formylmethionyl tRNA
37
IF1 and IF3 function
bind the 30S subunit to prevent premature binding to the 50S subunit
38
IF2 function
works in cooperation with GTP to deliver fMet-tRNAf to the mRNA (which is already correctly positioned on the 30S subunit by the Shine-Dalgarno sequence) → form the 30S initiation complex
39
What are the steps for initiation of translation in bacteria?
The 50S subunit binds → hydrolysis of GTP by IF2 → the initiation factors depart → forms the 70S initiation complex
The fMet-tRNAf occupies the P site bound to AUG of the ribosome → establishing the reading frame
FINISH LATER WHEN I HAVE TIJME
40
elongation factors EFs
a group of proteins that facilitate the addition of amino acids to the growing polypeptide chain during translation by interacting with the ribosome, tRNAs, and other molecules involved in protein synthesis
41
EF-Tu full name and function
Elongation Factor Thermo Unstable (the key elongation factor for bacterial translation)
function: binds to an aminoacyl-tRNA and guides it to the ribosome's A site (helps ensure that each amino acid is properly delivered to match the mRNA codon in the A site)
Ef-Tu binds GTP (guanosine triphosphate) and uses the energy from GTP hydrolysis to drive the delivery process: EF-Tu binds to the aminoacyl-tRNA and GTP --> forming a complex that is delivered to the ribosome’s A site
- Once the correct tRNA’s anticodon matches the mRNA codon in the A site, GTP is hydrolyzed to GDP --> this reaction causes EF-Tu to change shape and release the aminoacyl-tRNA and thus allowing peptide bond formation
- HOWEVER, if the codon-anticodon pairing is incorrect, GTP hydrolysis does NOT occur and EF-Tu does not release the tRNA, taking it back and preventing the wrong amino acid from being added
42
elongation description
The ribosome travels along the mRNA and reads each codon
Elongation factors deliver aminoacyl-tRNA to the ribosome
43
what are the steps of the GTP-GDP cycle of EF-Tu?
1. EF-Tu-GTP binds tRNA and delivers the tRNA to the A site of the ribosome
2. Correct codon recognition stimulates the GTPase activity of EF-Tu → leaves the ribosome in its GDP form
3. EF-Ts binds to EF-Tu-GDP
4. EF-Ts induces release of GDP → EF-Ts departs as another GTP and tRNA bind to EF-Tu-GTP and the complex is ready for another delivery to the ribosome
44
describe how elongation factors deliver aminoacyl-tRNA to the ribosome
- In the 70S initiation complex, fMet-tRNAf occupies the P site whereas the codon for the 2nd amino acid is exposed in the vacant A site
- Elongation factor Tu (EF-Tu) in association with GTP delivers the appropriate aminoacyl-tRNA as specified by the codon to the A site
--> If the anticodon pairs with the codon, GTP is hydrolyzed and EF-Tu departs (A site now occupied by the appropriate aminoacyl tRNA)
- Elongation factor Ts (EF-Ts): induces release of GDP from EF-Tu and GDP is replaced by GTP → another cycle can begin
45
does EF-Tu interact with fMet-tRNAf?
NO
EF-Tu does not interact with fMet-tRNAf because fMet-tRNAf is specifically involved in the INITIATION phase of translation and it is delivered to the ribosome by IF2, not by EF-Tu
EF-Tu is specialized for ELONGATION and its role is to deliver aminoacyl-tRNAs (not initiation tRNAs) to the ribosome AFTER the initiation step has been completed
46
where is the peptidyl transferase located and what is its function?
located within the 23S RNA of the large ribosomal subunit 50S and Peptidyl transferase catalyzes peptide-bond synthesis
1. The amino group of the aminoacyl-tRNA in the A site attacks the carbonyl group of the ester bond connecting the growing polypeptide chain to the peptidyl-tRNA in the P site
→ forms an 8-membered transition state
2. this transition state collapses to form the peptide bond and the peptidyl-tRNA in the P site is deacylated (aka it loses its attached amino acid, which is now a part of the growing polypeptide chain)
--> the deacylated tRNA is then released from the ribosome and the aminoacyl-tRNA in the A site now becomes the new peptidyl-tRNA in the P site
47
describe how some of the catalytic power is also due to catalysis by proximity and orientation
While the 23S rRNA catalyzes the actual chemical reaction, some of the catalytic power comes from the proximity and orientation of the reacting molecules
The ribosome ensures that the amino group of the incoming aminoacyl-tRNA and the carbonyl group of the peptidyl-tRNA are correctly positioned for the nucleophilic attack, which speeds up the reaction
The ribosome’s structure helps bring the molecules together in a precise alignment, which is essential for efficient and accurate peptide bond formation
48
why is the ribosome called a ribozyme?
ribosome is called a ribozyme because it exhibits catalytic activity that is driven primarily by RNA, rather than by proteins (contains a catalytic subunit/component)
49
what is the mechanism of protein synthesis/elongation (4 steps)
- The cycle begins with peptidyl-tRNA in the P site
1. An aminoacyl-tRNA binds in the A site
2. With both sites (A and P) occupied, a new peptide bond is formed
3. The tRNAs and the mRNA are translocated through the action of elongation factor G → moves the deacylated tRNA to the E site
4. tRNA is free to dissociate to complete the cycle
50
describe the translocation mechanism during elongation
translocation: the moving of the mRNA through the ribosome by one codon (3 nucleotides) and the process is powered by GTP hydrolysis
1. after peptide bond formation, the peptidyl-tRNA moves from A to P site and the uncharged tRNA (aka without the amino acid) moves from P to E site
2. the ribosome translocates along the mRNA by one codon and this process is powered by EF-G and the hydrolysis of GTP
3. the ribosome is left in a new state where the A site is vacant, the P site holds the peptidyl-tRNA, and the E site holds the uncharged tRNA
--> the process prepares the ribosome for the next round of elongation
51
what follows after the formation of a peptide bond?
The formation of a peptide bond is followed by the GTP-driven TRANSLOCATION of tRNAs and mRNA
52
Elongation factor G (aka translocase) function
a GTP-binding protein that binds to GTP and uses the energy from GTP hydrolysis to move the ribosome along the mRNA by one codon
--> this movement shifts the peptidyl-tRNA from A to P and the uncharged tRNA from P to E, effectively setting up the ribosome for the next elongation cycle
EF-G dissociates from the ribosome after GTP hydrolysis
53
what does it mean for a tRNA to be charged/uncharged?
charged tRNA: tRNA molecule that is linked to its corresponding amino acid (through a process called tRNA charging/aminoacylation)
- for a tRNA to be functional in protein synthesis, it must be "charged" with the correct amino acid that corresponds to the codon on the mRNA
54
Polyribosomes (polysomes)
Polyribosomes: the group of multiple ribosomes bound to an mRNA molecule and work together to translate a single mRNA molecule simultaneously
is found in both prokaryotes and eukaryotes
Have many ribosomes attached to the mRNA → all translating proteins at different stages
55
Operon
allows bacteria to simultaneously synthesize different proteins from a single mRNA molecule
- operon: a group of functionally related genes that can be transcribed together into a single mRNA molecule (they share a start site)
ie. Trp operon
eukaryotes: 5 separate genes over 4 different chromosomes --> transcribes 5 different/separate mRNA --> where each encodes for a different protein (5 proteins produced)
vs
prokaryotes: 1 operon (of 5 genes) --> transcribes 1 mRNA --> produces 5 proteins
56
Describe how termination of translation works (aka how does the synthesis of a polypeptide chain end when a stop codon is encountered?)
No tRNAs with anticodons complementary to the stop codons exist in normal cells
--> Release factors recognize the stop codons (UAA, UGA, or UAG) in the A site and stimulates the release of the completed protein from the tRNA in the P site
1. Elongation continues with the growing chain exiting the ribosome through the channel in the 50S subunit until a stop codon appears in the A site
2. RFs facilitate the attack of a water molecule on the ester linkage between the polypeptide chain and tRNA in the P site → releasing the complete protein
3. EF-G and ribosome release factor (RRF) catalyze the dissociation for the ribosome, mRNA, and attached tRNA in a reaction facilitated by GTP hydrolysis
57
Release factors RFs
the proteins that recognize the stop codons (UAA, UGA, or UAG)
--> A release factor recognizes a stop codon in the A site and stimulates the release of the completed protein from
- the tRNA in the P site
RFs interact with the peptidyl transferase
58
which stop codons do RF1 recognize?
UAA or UAG
59
which stop codons do RF2 recognize?
UAA or UGA
60
in eukaryotes, what do RF1 and RF2 resemble?
RF1 and RF2, in eukaryotes, resemble a tRNA molecule
61
RF3 (GTPase)
catalyzes the removal of RF1 or RF2 from the ribosome upon release of the newly synthesized protein
RF3 binds to GTP in its active form and then binds to the ribosome and promotes the hydrolysis of GTP --> induces a conformational change that helps release the RFs 1 and 2
- crucial for dismantling the translation complex and recycling and preparing the ribosome for the next round of translation
62
EF-G and ribosome release factor (RRF)
catalyze the dissociation for the ribosome, mRNA, and attached tRNA in a reaction facilitated by GTP hydrolysis
63
where does transcription and translation occur in eukaryotes?
transcription: nucleus (mRNA precursors are processed and splicing in the nucleus)
translation: cytoplasm (mRNA are transported to the cytoplasm for translation into protein)
64
where can ribosomes be located in the cytosol?
some are free in the cytosol and others are attached to membranes of the endoplasmic reticulum
65
describe how rRNAs are commonly designated
Ribosomal RNAs are commonly designated by their “S values”
→ refers to their RATE OF SEDIMENTATION in an ultracentrifuge
66
what are the components/subunits of a eukaryotic ribosome?
a large subunit 60S and a small subunit 40S
Bacterial ribosomes and eukaryotic ribosomes are similar in structure but eukaryotic ribosomes are larger
67
what gives ribosomes their shape?
ribosomal RNAs
68
in bacterial ribosome, what are the 2 key rRNAs that form the core of the large subunit 50S?
23S rRNA (the largest rRNA in the ribosome) and contains both the structural and catalytic roles
- contains the peptidyl transferase center
- acts as a ribozyme
5S rRNA: positioned in the large subunit and helps stabilize the structure
69
what is the L1 protein and what is its function?
L1: a protein found in the large subunit 50S of the ribosome
- located near the peptidyl transferase center and facilitates the exit of the deacylated tRNA from the P site of the ribosome
- the L1 protein protrudes from the surface of the ribosome and can be seen as a distinct protrusion in structural images --> serves as a REFERENCE POINT for the ribosome’s overall structure
70
how many proteins and rRNAs do each of the eukaryotic subunits contain?
2 subunits bond to form a complete eukaryotic ribosome (80S)
Large subunit 60S: 49 proteins & 3 rRNAs
Small subunit 40S: 33 proteins & 1 rRNA
71
how many different proteins and rRNA molecules does the complete ribosome contain in total?
the complete ribosome contains 82 different proteins & 4 different rRNA molecules
72
how many binding sites for an mRNA molecule and tRNAs does each ribosome have?
Each ribosome has 1 binding site for an mRNA molecule and 3 binding sites for tRNAs
The tRNA sites are designated A, P, and E
73
how is eukaryotic protein synthesis more complex than bacterial protein synthesis? (5 different ways)
1. Ribosomes: eukaryotic ribosomes are larger (40S and 60S subunits --> 80S ribosome) compared to bacterial ribosome (30S and 50S --> 70S)
2. Initiator tRNA: eukaryotic protein synthesis begins with a METHIONINE instead of formylmethionine like in E. Coli
- Met-tRNAi: a special initiator tRNA is required for eukaryotes
3. Initiation: the eukaryotic initiator codon is ALWAYS the FIRST AUG from the 5’ end of the mRNA
- eukaryotes require a larger number of initiation factors
4. Elongation and termination: similar but eukaryotes only have ONE release factor (eRF1) vs bacteria have TWO release factors (RF1, RF2)
5. Organization: eukaryotic protein synthesis occurs in the cytoplasm vs RNA synthesis occurs in the nucleus
- Eukaryotic protein synthesis machinery is organized into large complexes associated with the cytoskeleton
74
what is the difference between eukaryotes and prokaryotes regarding the start codon of translation?
In prokaryotes: the AUG is preceded by the Shine-Dalgarno sequence and formyl-Met-tRNA binds to the initiator codon
In eukaryotes: the AUG nearest the 5’ end is the initiator codon
Location of the initiator codon establishes the reading frame
75
what are the steps for the initiation of translation in eukaryotes?
1. Translation initiation factors bind to the initiator tRNA on the small ribosomal subunit
2. mRNA binds to the ribosome
3. The small ribosomal subunit with bound initiator tRNA moves along the mRNA strand searching for the first AUG
4. After the AUG codon has been found, translation initiation factors dissociate and the large ribosomal subunit binds to the small subunit
5. Charged tRNA binds to the 2nd codon
6. 1st peptide bond forms
76
what are the 4 steps of eukaryotic elongation?
1. A charged tRNA carrying the next amino acid to be added to the polypeptide chain binds to the vacant A site on the ribosome by forming base pairs with the mRNA codon
--> The A and P sites are sufficiently close together that their two tRNA molecules are forced to form base pairs with codons
2. The carboxyl end of the polypeptide chain (aka amino acid 3 in step 1) is uncoupled from the tRNA at the P site and joined by a peptide bond to the free amino group of the amino acid linked to the tRNA at the A site
--> This reaction is carried out by a catalytic site in the LARGE subunit
3. A shift of the large subunit relative to the small subunit moves the two bound tRNAs into the E and P sites of the large subunit
4. The small subunit moves exactly THREE nucleotides along the mRNA molecule → bringing it back to its original position relative to the large subunit
--> This movement ejects the spent tRNA and resets the ribosome with an empty A site so that the next charged tRNA molecule can bind
77
Do tRNAs recognize the stop codons UAA< UAG, UGA?
NO
Release factors are the ones that recognize the stop codons → RFs bind to the stop codons on the mRNA and signal the ribosome to terminate protein synthesis
78
Do the stop codons specify for amino acids?
NO
No tRNAs with anticodons complementary to the stop codons exist in normal cells
79
Mutations in initiation factor 2 (IF2) result in a which disease?
vanishing white matter (VWM) disease: characterized by the disappearance of brain nerve cells that are replaced by cerebrospinal fluid
normal brain: the MRI visualizes the white matter as dark gray
diseased brain: MRI reveals that the white matter is replaced by cerebrospinal fluid which is seen as white
80
how can the differences between eukaryotic and bacterial ribosomes be exploited for the development of ANTIBIOTICS?
The ability of many antibiotics to inhibit bacterial protein synthesis while leaving eukaryotic protein synthesis unaffected makes them powerful therapeutic agents
81
what are 2 examples of antibiotics and their effects on bacterial and eukaryotic protein synthesis?
Ie. Streptomycin: interferes with the binding of fMet-tRNA → inhibits protein synthesis initiation in BACTERIA
Ie. Puromycin: inhibits protein synthesis in BOTH eukaryotes and bacteria by releasing uncompleted polypeptide chains from the ribosome
- Resembles the aminoacyl terminus of an aminoacyl-tRNA
--> Its amino group joins the carboxyl group of the growing polypeptide chain to form peptidyl-puromycin that dissociates from the ribosome
- Peptidyl-puromycin is stable since puromycin has an amide (red) instead of an ester linkage
82
what are some antibiotic inhibitors of protein synthesis?
1. streptomycin and other aminoglycosides
2. tetracycline
3. chloramphenicol
4. cycloheximide
5. erythromycin
6. puromycin
83
streptomycin and other aminoglycosides effect
inhibit initiation and cause the misreading of mRNA (bacteria)
84
tetracycline effect
binds to the 30S subunit and inhibits the binding of aminoacyl-tRNAs (bacteria)
85
chloramphenicol effect
inhibits the peptidyl transferase activity of the 50S ribosomal subunit (bacteria)
86
cycloheximide effect
inhibits translocation (eukaryotes)
87
erythromycin effect
binds to the 50S subunit and inhibits translocation (bacteria)
88
puromycin effect
causes premature chain termination by acting as an analog of aminoacyl-tRNA (bacteria and eukaryotes)
89
what are 2 examples of toxins that modify 28S rRNA?
ricin
alpha-sarcin
90
ricin and its effects on protein synthesis
ricin: a small highly toxic protein found in castor beans
- Has a catalytic activity that cleaves adenine from a nucleotide in 28S rRNA that is crucial for binding elongation factors → HALTS protein synthesis
91
alpha-sarcin and its effects on protein synthesis
alpha-sarcin: a ribonuclease (not a glycoside hydrolase)
- Cleaves a single phosphodiester linkage in the 28S RNA in the same loop where ricin acts → this cleavage COMPLETELY inhibits protein synthesis
Researchers are attempting to modify alpha-sarcin for use as an antitumor drug
92
diphtheria toxin and its effect on protein synthesis in eukaryotes
Diphtheria toxin blocks protein synthesis in eukaryotes by inhibiting translocation
- The toxin covalently attaches ADP-ribose to an amino acid in EF2 and this modification prevents elongation --> halts protein synthesis
- In unimmunized individuals, infection can be fatal
93
Corynebacterium diphtheria
Corynebacterium diphtheria: the bacterial cause of diphtheria that grows in the upper respiratory tract of an infected individual → produces a toxin that inhibits protein synthesis
94
what do ricin, alpha-sarcin, and diphtheria toxin all have in common?
ricin, alpha-sarcin, and diphtheria toxin all act by INHIBITING protein synthesis ELONGATION
Ricin & alpha-sarcin: do this by covalently modifying rRNA
Diphtheria toxin: does this by covalently modifying the ELONGATION FACTOR
95
summarize the stages of translation (initiation, elongation, termination)
1. Initiator tRNA binds to the start codon (AUG)
2. Large ribosomal subunit binds
3. New tRNA enters the A-site of ribosome
4. The new tRNA moves to the P-site, the initial tRNA moves to the E-site
5. Peptide bond is formed between the amino acids
6. The tRNA at the E-site exits the ribosome
7. If the codon is a stop codon, a release factor binds and the polypeptide is released from the ribosome
96
compare the processes of DNA replication, transcription (RNA synthesis), and translation (protein synthesis)
process/directionality:
DNA: replication 5' --> 3'
RNA: transcription 5' --> 3'
protein: translation N --> C (amino --> carboxyl)
subunit:
DNA: dNTPs
RNA: NTPs (UTP not TTP)
protein: aa-tRNA (aminoacyl-tRNA synthetase)
enzymes:
DNA: DNA polymerase, RNA primase, ligase, repair...
RNA: RNA polymerase, etc.
protein: ribosome, etc.
start/stop
DNA: origin of replication/no stop (copies entire DNA strand)
RNA: promoter/terminator
protein: ribosome-binding site (start codon AUG)/stop codon UAA, UAG, UGA
