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157 Cards in this Set
- Front
- Back
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What occurs during translation?
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A tRNA adaptor "translates" the nucleotide sequence of an mRNA into the amino acid sequence of a polypeptide
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How was it determined that there must be three nucleotides to specify an amino acid?
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2 nucleotides provides 16 possible A.A. sequences; therefore need 3 nucleotides (64 possible sequences)
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What did Crick and Brenner determine from analyzing the effects of "combining" mutations that inserted or deleted one or more nucleotides?
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- Code is non-overlapping
- No punctuation in code - Each codon = 3n nucleotides |
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What is meant by cell-free protein synthesis? What was it used to understand?
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- Cytosol (ribosomes, tRNA, and enzymes) was placed in tubes
- ATP, GTP, and Poly(U) added - Radioactively labeled AA added individually to tubes so that only polypeptide containing the AA for UUU would be coded - Poly-phenylalanine created (Repeated for CCC and AAA) |
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How were poly(U), poly(C), and poly(A) prepared for the cell-free protein synthesis experiment?
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- Polynucleotide Phosphorylase catalyzes the reaction of
(NMP)n + NDP <=> (NMP)n+1 + Pi (generates long polynucleotides for amino acids to link to) |
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How did researchers determine the amino acid code of nucleotides for those other than UUU, or AAA, or CCC?
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Made 0.75:0.25 ratios of different nucleotides; i.e.:
75% U, 25% G generates UUU > UGU, GUU, UUG > UGG, GGU, GUG > GGG Phe > Leu, Cys, Val > Trp, Gly (does not actually establish sequence of codons, but know base composition) |
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How did Khorana clarify which codons were for which amino acids?
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He chemical synthesized polyribonucleotides with repeating patterns (ex: ACACACAC...); possible codons: ACA or CAC; amino acids were threonine and histidine, which using previous data (2A, 1C = histidine)...etc.
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What was the significance of the high Mg+ conditions for defining RNA sequences?
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With high Mg+ conditions, polypetides can be initiated even without a start codon; with low Mg+ conditions (realistic conditions), only could begin if AUG sequence was included (for Formyl-Methionine)
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What is a sequence of three adjacent nucleotides in a nucleic acid that code for a specific amino acid called?
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Codon
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What is the reading frame?
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A contiguous, non-overlapping set of three-nucleotide codons in DNA or RNA (no punctuation)
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What signals the beginning of translation in the genetic code?
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The initiation codon - AUG (Met)
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What signals the ending of translation in the genetic code?
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A Termination codon - UAA, UAG, UGA (also known as stop codons or nonsense codons) - signal ending of polypeptide synthesis and do not code for an amino acid
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What sets the reading frame?
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The initiation codon (AUG)
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What is it called when a reading frame does not contain a termination codon among 50 or more codons?
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Open Reading Frame (ORF) - usually corresponds to genes that encode proteins
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Why would programs search genetic code for stretches of DNA reading frames without a stop codon?
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These sections usually encode a gene (the typical protein with a molecular weight of 60,000 would require an ORF (open reading frame) with 500 or more codons)
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In what direction does translation along an RNA template proceed?
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5' --> 3'
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What is meant by calling the genetic code "degenerate"?
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- Does not suggest code is flawed
- Each amino acid may have two or more codons (but each codon only specifies for one amino acid) - Some AA have only one codon, some have up to six codons |
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What reads the mRNA codons? How?
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tRNA base pair with mRNA codons at a three base sequence on the tRNA called the anticodon
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What relationships did Crick propose in his Wobble Hypothesis?
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1. The first two bases of an mRNA codon form strong base pairs with the last two bases of tRNA anticodon
2. First base of anticodon (position 3 for codon) determines if tRNA can "read" 1, 2, or 3 codons (C or A can recognize one; U or G can recognize two; I can recognize three) 3. Codons that differ in either of the first two bases require different tRNAs 4. Minimum of 32 tRNAs are required to translate all 61 codons |
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Why are there 32 minimum tRNA anticodons and not just 31?
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The last one is for the initiation tRNA (two separate tRNA's for Methionine, one for formyl-Met)
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In the Wobble Hypothesis what can the different 5' bases of anticodon recognize on the 3' base of codon?
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5' anticodon (pos. 1) --> 3' codon (pos. 3)
C --> G A --> U U --> A or G G --> C or U I --> U, C, or A (U, G, and I can do non Watson-Crick base pairing) |
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What are the five stages of protein synthesis?
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1. Activation of Amino Acids
2. Initiation of Translation 3. Elongation 4. Termination and Release 5. Folding and Processing |
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What essential structural components are required during the activation of amino acids?
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- Amino Acids
- Aminoacyl-tRNA Synthetases - tRNAs - ATP - Mg2+ |
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What essential structural components are required during initiation of protein synthesis?
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- mRNA
- N-Formylmethionyl-tRNA^fMet - Initiation Codon on mRNA (AUG) - 30s Ribosomal Subunit - 50s Ribosomal Subunit - Initiation Factors (IF1, IF2, IF3) - GTP - Mg2+ |
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What essential structural components are required during elongation of protein synthesis?
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- Functional 70s Ribosome
- Aminoacyl-tRNAs - Elongation Factors (EF-Tu, EF-Ts, EF-G) - GTP - Mg2+ |
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What essential structural components are required during termination of protein synthesis?
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- Termination Codon in mRNA
- Release Factors (RF1, RF2, RF3, RRF) - EF-G - Mg2+ |
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What is the structure of the bacterial ribosome?
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Whole ribosome = 70S
Large subunit = 50S Small subunit = 30S |
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What is the structure of the eukaryotic ribosome?
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Whole ribosome = 80S
Large subunit = 60S Small Subunit = 40S |
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In the bacterial 70S ribosome, how many proteins are in each subunit? How many rRNAs?
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30S subunit: 21 proteins (S1-21), 1 rRNA (16S)
50S subunit: 36 proteins (L1-36), 2 rRNA (5S and 23S) |
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What percentage of the ribosome is RNA? What percent is protein?
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2/3 RNA
1/3 Protein |
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What is found at the catalytic center of the ribosome (where the peptide bonds are formed)?
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RNA, no protein nearby
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What defines the amino acid arm?
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Carries a specific amino acid esterified by its carboxyl group to the 2' or 3' hydroxyl group of the A residue at the 3' end of the tRNA
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What defines the anticodon arm?
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Contains the anticodon triplet; 5' end is the Wobble position
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What defines the D arm of the tRNA?
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Contains two or three D residues at different positions; important for overall folding of tRNA
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What defines the TΨC arm of the tRNA?
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Contains T residue usually not in RNAs and Ψ which has an unusual carbon-carbon bond; important for overall folding of tRNA; interacts with large subunit rRNA
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What occurs during the first step of protein synthesis?
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- Aminoacyl-tRNA synthetases (one for each AA) esterify the 20 amino acids to their corresponding tRNAs
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What is the aminoacyl-tRNA synthetase reaction mechanism?
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- AA attacks ATP at α-P to create aminoacyl adenylate (Aminoacyl-AMP), releases PPi (which is further hydrolyzed to 2Pi for energy release)
- Aminoacyl group is transferred to the 3'-OH of the tRNA's 3' Adenosine residue, either directly or by first going on the 2'-OH of A residue and transesterifying to 3'-OH (release AMP) |
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What is the overall reaction for amino acid activation?
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Amino Acid + tRNA + ATP --(Mg2+)--> aminoacyl-tRNA + AMP + 2Pi
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What are the two purposes of aminoacylation of tRNA?
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- Activates an amino acid for peptide bond formation
- Ensures appropriate placement of the amino acid in a growing polypeptide |
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What is an example of the proofreading capabilities of an aminoacyl-tRNA synthetase?
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- Ile-tRNA synthetase favors Isoleucine over Valine by factor of 200 (due to single methylene); error rate though is less than 1 in 3000
- Val-AMP fits the proofreading site on the synthetase for Isoleucine (because it has one less methylene) and is hydrolyzed (whereas Ile wouldn't fit, so won't be hydrolyzed) - Synthetases can also hydrolyze the ester linkage between amino acids and tRNAS (accelerated for incorrectly charged tRNAs) |
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Why is it okay that the overall error rate of protein synthesis is not as low as DNA replication?
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- Flaws in a protein are eliminated when it is degraded and are not passed on to future generations; less biological significance
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What is referred to by the "Second Genetic Code"?
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The interaction between aminoacyl-tRNA synthetases and tRNAs
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How do aminoacyl-tRNA synthetases recognize their respective tRNA targets?
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- There are identified nucleotide positions that are involved in discrimination by the aminoacyl-tRNA synthetases; concentrated in the amino acid arm and the anticodon arm, and other places
- May only be ten or more specific nucleotides required for recognition |
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What did the Benzer experiment do?
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- Correct Amino Acid was added to respective tRNA by synthetase (Cysteine)
- Chemical reduction switched AA to a different one (Alanine) - In vitro Alanine was incorporated into hemoglobin polypeptide at sites that normally contained Cysteine |
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What did the Benzer experiment prove?
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Ribosome does not proofread whether amino acid attached to the tRNA is correct; faithful translation depends on correct interaction between aminoacyl-tRNA synthetases and tRNAs
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How is the initiator fMet-tRNA synthesized vs. Met-tRNA?
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- Both tRNAs are first attached to methionine by Met-tRNA synthetase
- Next a transformylase transfers a formyl group from N10-Formyl-THF to the amino group of the Met residue - Transformylase is more selective than Met-tRNA synthetase so only attaches to tRNA-fMet |
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How did the experiment by Dintzis demonstrate that protein synthesis proceeds from the N-terminal to the C-terminal?
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- Reticulocytes (immature RBCs) were labeled with radioactive leucine
- Samples were isolated at various times afterwards - Only complete chains containing radioactive Leu after 4 minutes were already nearing completion when label was added - At later times successively longer segments were completed - Unlabeled end was defined as initiating end (amino terminus) |
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What is the Shine-Dalgarno sequence?
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- A sequence in an mRNA that is required for binding bacterial ribosomes
- Four to nine purine residues - Base pairs with a a complementary pyrimidine rich sequence near the 3' end of the 16S rRNA of 30S ribosome |
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What are the steps of initiation of protein synthesis?
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- 30S ribosomal subunit binds initiation factors IF-1 and IF-3
- IF-3 prevents 30S and 50S subunits from combining too soon - 5'AUG guided by Shine-Dalgarno sequence - IF-1 binds at the A site and prevents tRNA binding at this site during initiation - GTP-bound IF-2 and initiating fMet-tRNA bind directly to P site - Complex combines with 50S ribosomal subunit to form 70S ribosome (initiation complex) - GTP bound to IF-2 is hydrolyzed - IF's depart from ribosome |
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What is the initiation complex?
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A complex of a ribosome with an mRNA and the initiating Met-tRNA of fMet-tRNA, ready for the elongation steps
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What happens at the A site?
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Aminoacyl: site where incoming aminoacyl-tRNA's bind first (besides fMet-tRNA); IF-1 binds at A site during initiation
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What happens at the P site?
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Peptidyl: site where aminoacyl-tRNA's move to after leaving the A site
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What happens at the E site?
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Exit: site from which the "uncharged" tRNAs leave during elongation
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What is the function of the three Initiation Factors in bacteria?
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- IF-1 - prevents premature binding of tRNAS to A site
- IF-2 - facilitates binding of fMet-tRNA to 30S ribosomal subunit - IF-3 - binds to 30S subunit; prevents premature association of 50S subunit; enhances specificity of P site for fMet-tRNA |
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What are the steps of elongation during protein synthesis?
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- Appropriate aminoacyl-tRNA binds to a complex of GTP-bound EF-Tu at the A site
- GTP is hydrolyzed and EF-Tu and GDP are released (regenerated with GTP and EF-Ts) - AA at A site displaces tRNA in P site to form peptide bond; "uncharged" tRNA remains bound at P site - tRNAs then shift to span two different sites - Translocation occurs as ribosome moves one codon toward the 3' end of the mRNA (A moves to P; P moves to E where it is released) - Ready for next elongation cycle |
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What are the roles of the important elongation factors for protein synthesis?
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- EF-Tu - attached to incoming aminoacyl-tRNA; bound to GTP which hydrolyzes from tRNA when bound at A site
- EF-Ts - Binds to EF-Tu-GDP and is released when new molecule of GTP comes - EF-G (translocase) - mimics the structure of EF-Tu complexed with tRNA; binds to the A site and displaces the peptidyl-tRNA (moving it to the P site) |
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How does the slowly hydrolyzable GTP analog affect protein translation?
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- Binds and activates GTPase for longer than usual
- Reduces the rate of protein synthesis - Improves the fidelity by increasing the proofreading intervals |
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How does the structure of the slowly hydrolyzable GTP analog compare to regular GTP?
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On the γ-P there is a Sulfur instead of Oxygen (GTPγS)
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What is peptidyl transferase?
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Catalyzes the peptide bond formation; catalyzed by the 23S rRNA (ribozyme)
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What occurs during translocation?
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Final stage of the elongation cycle; ribosome moves one codon toward the 3' end of the mRNA; shifts the anticodon of the dipeptidyl-tRNA from A to P and deacylated tRNA from P to E (where it is released); requires EF-G (translocase) and hydrolysis of GTP
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What are the energetic requirements during elongation?
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- During the first step, when the aminoacyl-tRNA enters the A site GTP is hydrolyzed on EF-Tu (GTP put back with help from EF-Ts)
- During the third step, when translocation occurs, EF-G hydrolyzes GTP to enter the A site |
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What role does 23S rRNA play in peptide bond formation?
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The peptidyl transferase activity forming the peptide bond, comes from the 23S rRNA ribozyme
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How does EF-Tu act as a proofreading mechanism to check for the proper codon-anticodon pairing?
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- EF-Tu - GTP is attached to the entering aminoacyl-tRNA; the time it takes for GTP to hydrolyze to GDP + Pi is the intrinsic "timer" for proofreading
- EF-Tu-GDP must be released before transpeptidation rxn can occur - If AA-tRNA dissociates before EF-Tu-GDP (due to weak/incorrect codon-anticodon intxn) no transpeptidation rxn will occur |
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When does termination begin?
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When a termination codon enters the ribosomal A site; signaled termination codons in the mRNA (UAA, UAG, UGA) activate the release factors
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What steps happen during termination?
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- Termination factors / Release factors contribute to:
- hydrolysis of the terminal peptidyl tRNA bond - release of the free polypeptide and the last tRNA from the P site - dissociation of the 70S ribosome into 30S and 50S |
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What are the roles of each release factor?
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- RF-1 recognizes the termination codons UAG and UAA
- RF-2 recognizes the termination codons UGA and UAA - RF-1 or RF-2 bind at terminationc odon and induce peptidyl transferase to transfer the polypeptide to a water molecule - RF-3 is thought to release the ribosomal subunit (a GTP protein hydrolyzed during release) |
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Why is polypeptide biosynthesis so highly energetically expensive?
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~ 30-40 peptide bonds formed per second
- Specificity and speed of translation requires 4 high-energy phosphate bonds per peptide bond (2 bonds for AA activation; 1 bond for AA-tRNA binding (EF-Tu); 1 bond for translocation (EF-G)) |
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What is a polysome?
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A complex of an mRNA molecule and two or more ribosomes; allows for the highly efficient use of the mRNA
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How does puromycin affect translation?
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- Puromycin is an amino acyl-tRNA analog
- Binds at the ribosomal A site and participates in peptide bond formation - Does not engage in translocation and dissociates from the ribosome shortly after joining the peptide - Prematurely terminates polypeptide synthesis |
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What is the impact of tetracyclines?
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- They inhibit protein synthesis in bacteria by blocking the A site on the ribosome, preventing the binding of aminoacyl-tRNAs
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What is the impact of chloramphenicol?
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Inhibits protein synthesis by bacterial ribosomes by blocking peptidyl transfer on the large subunit; it does not affect the cytosolic protein synthesis in eukaryotes
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What is the impact of cycloheximide?
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Blocks the peptidyl transferase of 80S eukaryotic ribosomes but not that of 70S bacterial ribosomes
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How are chloramphenicol and cycloheximide similar?
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Both block peptidyl transferase, but chloramphenicol affects prokaryotic ribosomes and cycloheximide affects eukaryotic ribosomes
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What does streptomycin do?
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A basic trisaccharide, causes misreading of the genetic code in bacteria at relatively low concentrations and inhibits initiation at higher concentrations
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How does diphtheria toxin affect protein synthesis?
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Catalyzes the ADP-ribosylation of a diphthamide residue of eukaryotic elongation factor eEF2 (analogous to EF-G in prokaryotes), thereby inactivating it
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How does Ricin affect protein synthesis?
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Extremely toxic protein of the castor bean, it inactivates the 60S subunit of eukaryotic ribosomes by depurinating a specific adenosine in 23S rRNA
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What are some differences between eukaryotic and prokaryotic translation?
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- Bacterial mRNA is often polycistronic (multiple genes/proteins per mRNA); eukaryotic mRNA is monocistronic (one mRNA = one protein)
- Bacterial ribosomes are smaller (70S vs. 80S) - Bacteria use only 3 initiation factors vs. nine for euks - Translation control is rare in bacteria - Bacteria recognize Shine-Delgarno sequence for initiation; Euks scan mRNA from 5' end (m7G-Cap) until first AUG; 40S subunit binds m7G-Cap with help of initiation factors - Eukaryotic initiator Met-tRNA is not N-formylated |
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How does initiation begin in eukaryotes?
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-eIF-4F complex (of eIF-4A, eIF-4E and eIF-4G help bindbinds to mRNA m7G cap; helps to bind 40S subunit of ribosome to mRNA
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What does eIF-2 do for eukaryotic protein translation?
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Facilitates binding of initiating Met-tRNA to 40S ribosomal subunit
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How is eIF-2 regulated by heme during protein translation?
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- When high [Heme], heme-controlled inhibitor (HCI) is inactive, allowing eIF-2-GDP to replace GDP with GTP and aid in translation
- When low [Heme], HCI is active, eIF-2-GDP is phosphorylated and GEF blocks eIF-2 from returning to it's role in translation |
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How is eIF-2 regulated by interferon during protein translation?
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- Interferons are secreted proteins from virus-infected cells
- Can induce RNA-dependent protein kinase which phosphorylates eIF-2 (similar to Heme-Controlled Inhibitor (HCI)) enabling eIF-2 from doing its role in translation |
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What is an interferon? What are the three classes? How do they act?
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A secreted protein from virus-infected cells; triggers antiviral state in other cells
- 3 classes: leukocyte, fibroblast, and lymphocyte - Phosphorylates eIF-2 - Activates RNaseL which degrades all mRNA shutting down translation |
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What are molecular chaperones?
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Proteins that interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways or providing micro-environments in which folding can occur
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What is the heat shock response?
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A response to protect against stress-induced damage (e.g. heat, toxins); all cells produce a set of conserved "heat shock proteins" (Hsps) which are chaperones; not just present during stress, many are constitutively expressed (Hsc)
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What are non-native states of proteins? When do they occur?
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- When proteins have exposed hydrophobic patches
- Occurs during protein translation, protein translocation into organelles, assembly of multi-subunit complexes, stress-induced damage |
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What is the function of Hsp70s?
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- Use energy from ATP hydrolysis
- Bind to regions of unfolded polypeptides that are rich in hydrophobic residues, preventing inappropriate aggregation - Some are induced by stress and some are constitutively expressed; interact with most newly synthesized proteins - Some block the folding of certain proteins that must remain unfolded until they have been translocated across a membrane |
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What is the structure of chaperonins?
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Made of two families: Hsp60s (GroEL) and Hsp10s (GroES)
- GroEL - 2 heptameric rings - GroES - 1 heptameric ring which sits on top of GroEL like a cap |
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How do chaperonins assist in folding?
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- An unfolded protein enters GroEL
- 7 ATP bind (one for each subunit of GroEL) and lead to conformation change of GroEL and binding of GroES - 7 Pi released while unfolded protein enters aqueous chamber and folds properly - 7 ATP hydrolyzed and GroES cap is released along with folded protein |
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What is the function of protein disulfide isomerase?
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Catalyzes the interchange of disulfide bonds until the bonds of the native conformation are formed
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What is the function of cis/trans prolylisomerase?
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Catalyzes the interconversion of the cis and trans isomers of Pro residue peptide bonds which can be a slow step in the folding of proteins with Pro in cis conformation
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What are prions?
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Proteinaceous Infectious Particles
- protein which is an infectious agent - no nucleic acid - highly stable |
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What is the cause of "Mad-Cow Disease" / Bovine Spongiform Encephalopathy?
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Prion
- misfolded brain protein - illness only occurs when the normal cellular protein occurs in an altered conformation - the misfolded protein converts other normally folded proteins to the disease-causing form - misfolded proteins aggregate to form amyloid plaques |
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What is the function of a signal sequence?
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- Directs a protein to its appropriate location in the cell
- Removed during transport or after the protein has reached its final destination |
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How does the position of the signal sequence affect the function of the signal?
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- N-Terminal sequences - import into ER or mitochondria
- C-Terminal sequences - retention in lumen of ER - Internal sequences - import into nucleus and peroxisomes |
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What is the targeting pathway for directing new proteins to the endoplasmic reticulum?
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1. Initiation of protein synthesis on free ribosomes
2. Signal sequence appears early in the process because it's on the N-terminus 3. Signal sequence and ribosome are bound by the large Signal Recognition Particle (SRP) which binds GTP to halt elongation 4. GTP-bound SRP directs the ribosome (with mRNA and polypeptide) to GTP-bound SRP receptors (on cytosolic face of ER); new polypeptide delivered to a peptide translocation complex (Sec 61 channel/translocon) in ER 5. SRP dissociates from ribosome; GTP is hydrolyzed on both SRP and receptor 6. Elongation of polypeptide resumes 7. Signal sequence is removed by a signal peptidase within the ER lumen 8. Ribosome dissociates and is recycled |
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What is the function of the Signal Recognition Particle (SRP)?
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Binds to the signal sequence and ribosome; interacts with SRP-receptor (SR) to guide protein/signal sequence to ER translocon
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What is the function of the ER translocon / peptide translocation complex?
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Opens when ribosome bound; closed when ribosome free; when open nascent polypeptide is delivered through into ER lumen
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Are signal sequences always removed?
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No, although for many they are removed during transport or after the protein has reached its final destination
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What is the role of the protein conduction channel ("translocon")?
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A large, gated channel that permits the entry of nascent polypeptides into the ER lumen and also opens laterally to permit the insertion of the hydrophobic membrane-spanning domains of the integral membrane proteins into the lipid bilayer of the ER
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What is the role of BiP in secretory protein synthesis?
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BiP (Binding Protein) is an Hsp70 chaperone in the ER lumen which coordinates with the polypeptide as it enters; BiP uses a grab and pull mechanism to bring the polypeptide into the lumen (post-translationally, using ATP hydrolysis) through the translocon
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What is the role of PDI in secretory protein synthesis?
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PDI (protein disulfide isomerase) arranges disulfide bonds necessary for proper folding of proteins (ex: inusulin)
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How is eukaryotic ER protein translocation similar/different to prokaryotic protein translocation?
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- Prokaryotes - no ER, so transported to periplasmic space between membranes or to extracellular medium
- Prokaryotes use SecB, SecA, SecYEG in transport, not SRP, SRP-Receptor, and Sec-61 translocon (but similar mechanism) - Prok: Leader peptides similar to ER signal sequences |
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How are proteins exported in bacteria?
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1. Newly translated protein binds to SecB
2. SecB delivers protein to SecA on membrane (associated with translocation complex SecYEG) 3. SecB released; SecA inserts into membrane; polypeptide enters translocation complex 4. ATP hydrolyzed; conformation change of SecA releasing protein 5. SecA binds ATP and pushes protein through more 4&5. repeat 6. Entire protein is in periplasm |
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Evolutionarily, how is protein translocation of bacteria similar to ER protein import of eukaryotes?
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- Plasma membrane (containing DNA and ribosomes) of ancient prokaryotes was invaginated to form a mesosome (gain greater surface area)
- Over time this invaginated membrane pinched off from outer membrane to form an inner and outer nuclear envelope which is attached to the endoplasmic reticulum - Processes that occur on prokaryotic plasma membrane related to eukaryotic processes of ER lumen |
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How does mitochondrial post-translation protein translocation occur?
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- Ribosome synthesizes protein completely (post-translational import); contains one signal sequence
- Hsp70 chaperones grab onto the protein and direct it towards a translocation complex (ATP hydrolysis required) on the outer membrane - Signal sequence and peptide continue through to second translocation protein on inner membrane (requires electrochemical gradient) - Hsp70 grabs from matrix to continue pulling peptide through second complex - Signal sequence is cleaved inside matrix |
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How does chloroplast post-translation protein translocation occur?
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- Two signal sequences (one to get through inner and outer membrane, the other to enter thylakoid)
- Peptide translocated across inner and outer membranes (using ATP hydrolysis for both) - Hsp70 grabs from inside to pull in - Signal sequence 1 cleaved - Peptide goes across translocation complex of thylakoid with signal sequence 2 cleaved inside |
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How are the soluble chaperones BiP and PDI retained within the ER?
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- They are soluble ER-resident proteins with C-terminal KDEL peptide
- a KDEL-receptor binds escaped ER-resident proteins in Golgi and transfers them back to the ER |
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What is the purpose of the KDEL peptide?
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- Important for retention/retrieval of proteins localized in ER (e.g. BiP and PDI)
- KDEL-receptors are in Golgi and return any escaped KDEL peptides |
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What is cotranslational protein targeting?
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Proteins with an N-terminal signal sequence can be translocated while translation is going on because the N-terminus is synthesized first (such as import into the ER)
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What is posttranslational protein targeting?
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Proteins with a C-terminal sequence can be translocated after translation is complete (such as for mitochondria and chloroplast)
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How are secretory vesicles formed?
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- Secretory proteins aggregate in the trans face of the Golgi
- Vesicles bud from trans Golgi; Clathrin-coats begin forming around immature secretory vesicle (GTP hydrolysis) - Excess membrane is removed with clathrin coat - Mature secretory vesicle is uncoated as GTP-->GDP (with concentrated protein within) |
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What are the possible destinations of a mature secretory cell?
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- Lysosome (clathrin coat)
- Regulated secretion - Constitutive secretion |
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What is the purpose of coat proteins?
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Coat-proteins, like clathrin, are used to build small vesicles in order to safely transport molecules between cells; used during bud/vesicle formation
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What are the different proteins involved in a secretory vesicle fusing with the plasma membrane of a neuronal cell? What are their roles?
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- Rab (GTPase required for targeting and fusion)
- Tether - SNAREs (SNAP receptors) (integral membrane proteins on vesicle [v-] and target [t-] membranes) - SNAP25 (soluble NSF attachment factor; adapter that mediates NSF binding to SNAREs) - NSF (NEM-sensitive factor, large chaperone required for disassembly of SNARE complexes) |
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What occurs as a secretory vesicle fuses with the plasma membrane?
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- Rab protein binds to transport vesicle for targeting and fusion (GTP bound)
- v-SNARE and t-SNARE bind to each other near Tether which helps dock vesicle - v-SNARE and t-SNARE tightly pull together forming cis-SNARE complex (4-helix complex), causing the membranes to fuse - Pore widens and contents are released |
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What is the function of protein coats?
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Budding and pinching off of vesicles from plasma membrane; recruits cargo proteins and targeting proteins SNAREs into budding vesicle; pinching off via dynamin
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What is the purpose of the nuclear pore complex?
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- Regulates bi-directional movement of molecules between cytosol and nucleoplasm
- Big enough for a ribosome |
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What passes through nuclear pores passively?
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Ions and small molecules
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What passes through nuclear pores, dependent on energy?
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RNAs/RNPs, Proteins, snRNPs
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What is found on proteins which are imported into the nucleus via nuclear protein transport?
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NLS (nuclear localization signal) - not cleaved; soluble receptor proteins recognize NLS
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What soluble receptor proteins recognize NLS on proteins imported into the nucleus?
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Importins, Karyopherins, Kaps
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What is the structure like of nuclear pores?
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Embedded in nuclear envelope with a spoke-ring-complex with cytoplasmic filaments on one side; nuclear basket on the other; central plug in the middle
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How does the nuclear localization sequence (NLS) interact with the nuclear pore complex?
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The NLS on the protein is recognized by an importin receptor (α subunit); this interaction leads to attachment to the cytoplasmic filaments of the nuclear pore
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How does the G-protein Ran confer directionality in transport across nuclear pore?
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- cytosol, Ran-GDP; nucleus, Ran-GTP predominate
- Ran-GDP favors association of cargo with importin - Ran-GTP favors disassociation of cargo from importin - Asymmetric distribution of Ran-GTP/GDP drives import |
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What are the main purposes of modifying proteins?
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- Regulation of activity: on/off
- Protein-protein interaction - Subcellular localization (targeting signals; membrane anchors) - Stability/aging (degradation) |
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What are the two ways in which carbohydrates can be attached as side chains to proteins?
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- N-linked - Asn residue (to amide on end of side-chain)
- O-linked - Ser or Thr residues (to -OH of side-chain) |
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What is the purpose of dolichol-phosphate?
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It is a molecule found on the lumenal side of the spanning through the membrane of the ER, the Phosphate sticking into the cytosol is the base for the oligosaccharide to build upon; from here the oligosac. is transferred to the Asn residue to form an N-glycosylated protein
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What does tunicamycin do?
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Mimics UDP-N-acetylglucosamine, therefore inhibiting the first step of the synthesis of the core oligosaccharide on Dolichol-P
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What is the pathway for synthesis and addition of N-linked glycans to secretory proteins?
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- Dolichol-P (on lumenal side; sugar begins building on cytosolic side)
- Halfway through synthesis, the molecule is translocated so the oligosach. is in the lumen - Co-translationally, the complete "core" oligosaccharide is attached to an Asn residue on the protein as it enters the lumen |
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How are "high-mannose" glycans processed?
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- Begins in ER with the removal of glucose residues and continues in Golgi
- It is generated by a trimming of the original mannose-rich oligosaccharide that leaves most of the mannose residues with no subsequent addition of further sugars |
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How are complex glycans formed?
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After Mannose residues are trimmed down to a core of 5 sugars, galactose, fucose, and sialic acid residues are added
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How is glycoprotein processing dependent upon secretory protein transport?
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- When proteins are suitably modified in the lumen of the ER they can be secreted to the Golgi
- In the Golgi, they are further modified and sorted so they may be sent to their final destinations |
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How is Mannose-6-P utilized for lysosomal protein sorting?
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Mannose-6P added to lysosomal hydrolases in cis-Golgi
- Man-6P receptors in trans-Golgi - Receptors sort proteins into vesicles destined for lysosome |
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What are GPIs?
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Glycosylated derivatives of phosphatidylinositol; GPI-linked proteins are integral proteins because they are tightly bound by hydrophobic interactions
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What is the purpose of having membrane proteins covalently linked to lipids (GPI)?
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The attached lipid provides a hydrophobic anchor that inserts into the lipid bilayer and holds the protein at the membrane surface
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What are the different types of protein lipid modification?
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- N-terminal myristoylation (C14 fatty acid)
- C-terminal isoprenylation - GPI anchoring |
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What is the general structure of the GPI anchor?
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Multiple attached lipid moieties are inserted into the lipid bilayer to anchor the protein at the surface (lipids attached to phosphatidylinositol which attaches to sugars which attaches to protein)
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How is the GPI anchor transferred to a protein in the ER?
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Protein with GPI-anchoring signal sequence (pro-protein) is attached to the GPI anchor in ER membrane by transamidase
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What locations of the body is collagen found in?
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- Cartilage
- Bone - Tendon - Artery - Skin |
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What is the role of collagen in the body?
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Extracellular glue; holds tissues together - evolved to provide strength (cross-links)
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What is the unique composition, sequence, and supermolecular structure of collagen?
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-Gly-Pro-Hydroxypro-Gly-Pro-Hydroxypro-
- Glycine residue fits nicely in the middle of the helical structure - Hydroxyproline is a post-translationally modified Pro residue - Three-stranded helix (α chains, not α helices) which are tightly wound and extensively cross-linked |
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What distinguishes one type of collagen from another?
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Different subunits / helices
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How is the thermostability of Collagen affected by hydroxyproline?
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Without hydroxyproline, collagen would denature/melt at a much lower temperature (below body temp); whereas hydroxyproline makes collagen more stable at higher temps
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What is the role of prolyl-hydroxylase?
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Enzyme (vitamin C cofactor) which converts proline + O2 --> hydroxyproline + CO2
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What are the post-translational modifications of collagen and where do they take place?
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- O-glycosylation
- N-glycosylation (high mannose glycans) - Prolyl-hydroxylase converts proline residues to hydroxyproline - Disulfide bond formation (protein disulfide isomerase, Hsp47 chaperone) |
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What is the role of protein degradation?
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Prevents buildup of abnormal or unwanted proteins and permits the recycling of amino acids; eliminates abnormal proteins and regulates cellular metabolism
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What is the purpose of ubiquitin?
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A small, highly conserved protein that targets an intracellular protein for degradation by proteasomes; several ubiquitin molecules are covalently attached in tandem to a Lys residue in the target protein by a specific ubiquitinating enzyme
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How does Ubiquitin get added to proteins?
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- Ubiquitin and E1 enzyme form a thioester linkage (+ ATP)
- E2 enzyme replaces E2 with new thioester linkage - In presence of E3, target protein replaces E2 with amide linkage on Lysine - Repeated cycles for additional ubiquitin attachments |
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How are ubiquitinated proteins degraded?
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By a large complex called the 26S proteasome
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What are the three different enzymes involved in ubiquitin-mediated protein degradation?
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- E1-SH = Ubiquitin activating enzyme (requires ATP)
- E2-SH = Ubiquitin conjugating enzyme (replaces E1 on Ub) - E3 = Ubiquitin-protein ligase |
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What are the two possible ubiquitin ligases? What are their functions?
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- HECT = Ub transferred from E2 to ER and then to substrate
- RING/U-box = Ub transferred directly from E2 to substrate |
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What is the structure of the 26S proteasome?
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- Two 19S caps on either end of a 20S core
- Peptidases are found on inner surface of 20S core, degrade substrate - 19S caps are made from a base and a lid; contain chaperones that unfold substrate and send it to 20S core |
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What is the role of the 26S proteasome?
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- Unfold substrate (19S caps w/ chaperones)
- Degrade substrate (20S core w/ peptidases) |
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What are the different functions that are known for poly-ubiquinated proteins?
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- Lys-48 - degradation (with proteasome)
- Lys-63 - DNA repair or endocytose protein and lysed at lysosome |
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What is the N-end rule?
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N-terminal amino acids are important determinants of protein half-life; Certain residues at the N-terminus cause the protein to be stabilized (>20hrs), destabilized (10-30min), or highly destabilized (2-3min)
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What is the role of SUMO?
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SUMO = Small Ub-related MOdifier (ubiquitin-related protein)
- required for protein targeting and for protection against degradation |
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What are some examples of diseases of protein degradation?
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- Cancer - oncogenic strains of HPV inactivate the tumor suppressor protein, p53; viral E6 protein binds cellular E6-associated protein, functions as a p53 ubiquitin ligase
- Neurodegenerative diseases (Alzheimer's, Parkinson's, Lou Gehrig's, Huntington's) - associated with Ub and proteasome |
