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What is the start codon?
AUG; it codes for methionine, and sets the reading frame for translation from the mRNA
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What is a point mutation, and what can result from a point mutation?
One base is replaced by another; when there is no change in the AA produced it is called a silent mutation; if one AA is replaced by another it is called a missense mutation; when the code for one AA is replaced by a stop codon it is called a nonsense mutation
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What is an insertion, and what can result from said insertion?
One or more bases are added to the sequence; if added in multiples of three AAs are added, when not added in multiples of three, it results in a frame shift mutation
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What is a deletion, and what can result from said deletion?
One or more bases are deleted; effects are similar to that of insertion
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What components are required for protein synthesis?
Amino acids, tRNA, mRNA, rsomes, aminoacyl-tRNA synthetases, protein factors, ATP, and GTP
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What does tRNA do?
Carries AAs to the site of protein synthesis
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What does mRNA do?
Carries genetic information from genes to the cytoplasm for translation
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What is a polyribosome?
Also known as a polysome; clusters of rsomes translating a single mRNA
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What do the small and large rsome subunits do?
The small subunit binds the mRNA and tRNAs and the large subunit catalyzes peptide bond formation
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What are the major roles of rsomes in translation?
It enables the initiation at the correct start site in prokaryotes and proofreads the correct codon-anticodon base pairing; ensures that each successive codon in mRNA engages precisely with the anticodon and does not slip by a nucleotide; it catalyzes the formation of the peptide bond
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How many binding sites are there for RNA molecules on a rsome?
There are three for tRNAs and one for mRNA
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What are the binding sites for tRNA?
- Aminoacyl-tRNA-binding site (A-site); binds the incoming tRNA molecule linked to an AA
- Peptidyl-tRNA-binding site (P-site); binds tRNA molecule that is linked to the growing peptide chain
- “Exiting”-tRNA-binding site (E-site); binds tRNA molecule that is not “free”
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What are the steps involved in protein synthesis?
- 1. AA activation, which involves the charging of a tRNA w/ its cognate AA
- 2. Initiation, which is the assembly of the
- components of the translational machinery before peptide bond formation
- 3. Elongation, which is the addition of AAs
- 4. Termination, which is the disassembly of the translational machinery and the release of the pp chain
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How does activation of AAs occur?
Activation is catalyzed by specific aminoacyl-tRNA synthetases; each aminoacyl tRNA synthetase attaches a single AA to all of the cognate tRNAs; AA + tRNA + ATP → Aminoacyl-tRNA + AMP + 2Pi
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In what direction does a pp grow?
N-terminus to C-terminus direction; the mRNA is read in the 5’ to 3’ direction
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What is the first codon at which translation is initiated?
AUG; which codes for Met; initiator tRNA recognizes the initiation codon; this is different from the tRNA that incorporates Met (tRNAf-Met in prokaryotes and tRNAMeti in eukaryotes)
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What is the Shine-Dalgarno sequence?
Found in prokaryotes, this is a region of mRNA that is upstream of the initiation codon, and it base pairs with the 3’ end of the 16S rRNA; it positions the start codon in the P-site of the rsome; it is found before the first AUG of each cistron in the polycistronic mRNA
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What is the Kozak consensus sequence?
The sequence is PuCCAUGG, where Pu is the purine nucleotide A or G; this is recognized by the ribosome as the translational start site; the sequence aids in defining the initial AUG codon for translation, the loss of which reduces the efficiency of translational initiation
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What association starts protein synthesis in prokaryotes?
The association of mRNA, f-Met‐tRNAf‐Met, and the ribosomal subunits
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What is prokaryotic initiation mediated by?
By a series of protein factors called initiation factors (IFs); there are three prokaryotic initiation factors which are called IF-1, IF-2, and IF-3
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How does initiation in prokaryotes occur (step-by-step)?
- 1. IF-3 binds the 30S rsome subunit, preventing it from prematurely complexing with the 50S subunit; IF-1 assists in this endeavor
- 2. IF-2 complexed to GTP (IF2-GTP) binds f-Met‐tRNAf‐Met and helps it to dock with 30S
- 3. As the mRNA binds, IF-3 helps to correctly position the complex such that the f-Met‐tRNAf‐Met interacts via base pairing with AUG at the P-site
- 4. As 50S joins the complex, IF2-GTP is hydrolyzed, and 50S serves as the GTPase activating protein for IF-2
- 5. A complex consisting of the 70S rsome, mRNA, and f-Met‐tRNAf‐Met base paired with mRNA at the P-site will be formed; there will be no more Ifs or GTP or GDP bound to it
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How does initiation in eukaryotes occur (step-by-step)?
- 1. A pre-initiation complex composed of several initiation factors, small rsome subunit, initiator tRNA, and Met-tRNAMeti is formed
- 2. The pre-initiation complex binds to the mRNA at the 5’ cap, and translocates along the mRNA in the 5ʹ → 3′ direction by a process called scanning, until the initiation codon is reached; this is facilitated by eIF4A
- 3. GTP bound to eIF2 is hydrolyzed as the large ribosomal subunit joins the complex
- 4. A complex consisting of the complete 80S ribosome, mRNA, and Met-‐tRNAMeti base paired with mRNA at the P‐site is formed
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How does elongation in prokaryotes/eukaryotes occur (step-by-step)?
- 1. EF-Tu/eEF1A-GTP binds and delivers an
- aminoacyl-tRNA to the A-site
- 2. The interaction of EF-Tu/eEF1A-GTP-aminoacyl-tRNA with the rsome hydrolyzes the GTP on EF-Tu/eEF1A-GTP-aminoacyl-tRNA to GDP + Pi, EF-Tu/eEF1A undergoes a large conformational change and the complex dissociates leaving the aminoacyl-tRNA at the A-site with the amino group of the AA near the 3’ end of the peptidyl tRNA
- 3. The ribozyme 23S rRNA catalyzes peptide bond formation between the amino N of the AA linked to the tRNA in the A-site to the
- carbonyl C of the AA in ester linkage to the tRNA in the P-site (this one loses
- it’s tRNA)
- 4. Once the peptide bond formation is complete, the E-site is empty, the P-site is occupied by the tRNA that unloaded the peptide, and the A-site is occupied by the tRNA which is attached to the nascent PP
- 5. EF-G/eEF2 is activated through its interaction with the rsome, and hydrolyzes its bound GTP to GDP + Pi; this causes EF-G/eEF2 to dissociate from the rsome
- 6. Step 5 cases the tRNA attached to the nascent PP to be pushed from the A-site to the P-site, and the empty tRNA that was in the P-site shifts to the E-site
- 7. The A-site is now open for the next EF-Tu/eEF1A-GTP-aminoacyl-tRNA to come and base pair with the next codon
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How does EF-Tu/eEF1A-GTP regenerate?
EF-T/eEF1B functions to reactivate eEF1A; it causes eEF1A to release its bound GDP; when eEF1A dissociates from eEF1B, then eEF1A binds a fresh GTP
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How does termination in prokaryotes and eukaryotes occur?
Protein synthesis is terminated when the translocation reaction brings a stop codon to the A site of the ribosome; this requires the release factors RF/eRF (prokaryotes/eukaryotes); they allow the nascent PP chain to be released
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How is the nascent PP chain released from the rsome during termination?
The release factors recognize stop codons and occupy the A site; this changes the specificity of peptidyl transferase such that the growing peptide chain is transferred from the peptidyl--‐tRNA to H2O, thus releasing the nascent peptide chain
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How does the rsome begin a new round of translation?
After releasing the nascent PP and the release factors the rsome is still bound to the mRNA and two “empty” tRNAs in the E and P-sites; to begin translating again a ribosomal recycling factor (RRF in prokaryotes and eRRF in eukaryotes) is required to release the tRNAs and mRNA from the rsome and allow the rsome to separate into subunits; this separation is required to begin translating again
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How do viruses promote self-replication?
Viruses shut off translation of host mRNA and promote the translation of viral RNA; they code for proteins that either inhibit protein‐protein interaction among the various proteins involved in mRNA cap-dependent translation initiation or degrade the eukaryotic initiation factors
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How does the poliovirus promote self-replication?
It has a protease which specifically cuts eIF4G and inhibits translation initiation; the viral mRNA is translated b/c it has an internal rsome entry site (IRES) where the rsome can bind and start translating viral mRNA
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Action of various antibiotics
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What is a hydrophobic signal sequence?
It is found in proteins destined for the ER, lysosomes, secretion, or membranes; this is a sequence in the approximately first 20 AA residues, which allows that region of the protein molecule to pass through the plasma membrane; in proteins this hydrophobic signal is retained and helps in anchoring the protein to the membrane; in secretory proteins, the N‐terminal signal sequence is cleaved and the protein is released into the lumen of the ER; glycosylation and packaging of the proteins into vesicles takes place in the Golgi
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What regulation of what cellular processes is controlled by the ubiquitin-proteasome pathway?
Degradation of misfolded proteins and cell cycle regulators; main mechanism for catabolism of proteins; closely tied to the functionalities of the cell cycle, gene expression, immune and/or stress response, and apoptosis
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What are the two major steps of the ubiquitin-proteasome pathway?
Conjugation (the process of targeting a substrate protein with several ubiquitin molecules) and degradation
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How does conjugation in the ubiquitin-proteasome pathway occur?
ATP dependent; the first Ub is attached to the Lys residue on the target protein; addition Ub molecules are attached to the previous Ub molecule to form a chain
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How does degradation in the ubiquitin-proteasome pathway occur?
The ubiquinated substrate protein enters the proteasome, which is a hollow, cylindrical complex with 3-7 protease active sites on the interior; the complex binds to the polyUb proteins and regulatory proteins in the complex release the Ubs for reuse; the protein is unfolded using the energy of ATP, and is then translocated into the main chamber; the protease sites cleave the peptide at specific points producing a set of peptides which are ~8 AAs long
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What can dysregulation of the ubiquitin-proteasome pathway contribute to?
Several neural diseases; may lead to brain tumors such as astrocytomas
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What does underactivity of proteasomes result in?
Results in the accumulation of proteins and plaque buildup, leading to neurodegenerative diseases such as Alzheimer's, Parkinson’s and Huntington’s diseases
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What does inhibition of the proteasomal pathway lead to?
Results in the arrest of cell cycle progression; this is being utilized to kill cancer cells
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