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84 Cards in this Set
- Front
- Back
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General definition of tRNA
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An RNA molecule that serves an adaptor in translation. At the one, it decodes the mRNA and at the other end, it carries the appropriate amino acid sequence.
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Triplet Code
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For 20 amino acids, at least 3 nucleotides are necessary per codon.
4^(3 bases) = 64 possible # of AA |
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What would happened if you an overlapping code?
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If you it were overlapping, a single nucleotide change would change 3 amino acids.
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How do we know that the code in non-overlapping?
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Because a single nucleotide results in the change of one amino acid.
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Frame shit
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Deletion or insertion of base results in complete change of the code downstream since the reading frame has changed.
Often leads to truncation because of stop codons. |
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What are two experiments used to decipher the code?
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1. Use trinucleotides to bind charged tRNA to ribosomes and see amino acids where translated. Look at the relative increase in labeled each type of aminoacyle-tRNA bound to ribosomes.
2. Use cell-free system and defined polyribonucleotides. |
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Why is there more than one polypeptide product in using the cell-free translation system and defined polynucelotides?
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All three reading frames are use to decode the polynucelotide.
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Stop Codons
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UAA, UAG, UGA
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Start Codon
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Met- AUG
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Degeneracy
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Some amino acids are specified by one codon, while many are specified by multiple codons.
This occurs because there are 64 possible codons and only 20 amino acids. |
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Why degeneracy advantageous?
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It can minimize the deleterious effects of mutations since the 3rd poisition mutation can be silent.
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How is degeneracy achieved?
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Wobble-Pairing: the first position in the anticodon that pairs with the third position in the codon pairs more loosely.
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Inosine
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Purine base that pairs with A, U and C.
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How many codons can be recognized when the 5' base of the anticodon is C or A?
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One
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How many codons can be recognized when the 5' base of the anticodon is U or G?
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Two, because U can pair with A or G and G can pair with C or U.
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How many codons can be recognized when the 5' base of the anticodon is I?
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Three because I can bind to eith A, U or C.
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Why is base pairing not as rigid with translation?
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No helix is made.
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Where is inosine always found?
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In the 5' base of the anticodon.
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What happens when codons differ in either of the the 1st two bases?
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The codons require different aminoacyl-tRNAs.
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What is the min number of tRNAs required to translate all 61 codons?
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32 tRNAs
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What is needed for the translation machinery?
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mRNA
tRNA ribosomes soluble protein factors for inititation, elongation and termination. |
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Where is the amino acid attached to tRNA?
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3' adenosine residue
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What forms the L structure?
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The attachment of the L and TC arm.
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How are bacterial and eukaryotic ribosomes?
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The eukaryotic ribosomes are larger.
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Where are the proteins located in the ribosome?
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The protein are located on the outside, away from where translation occurs (catalytic center).
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How do most anitbiotics work?
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They interfere with protein synthesis by targeting ribosomal RNA.
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Where does the interaction of tRNA and ribosome occur?
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3' acceptor end of the tRNA with a conserved region on 23S rRNA.
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Colicin E3
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Abolishes protein synthesis by cleaving a single bond in 16S rRNA.
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What is essential for peptidyl transferase activity?
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23 rRNA
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In the the ribosome, what positions the start site in mRNA?
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A sequence in the 3' end of 16S rRNA establishes the correct reading frame by position in the AUG in the right place.
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Why are SD-16s interaction required for protein synthesis in prokaryotes but not eukaryote?
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Prokaryotes are polycistronic and new translation unit needs start multiple points.
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In what direction is the mRNA read?
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5' to 3'
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In what direction is the protein produced?
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N to C
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How is the direction of protein synthesis determined?
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Isolate ribosomes already engaged in translation and added a radio label.
When he stopped translation early, he noticed that all the label where in the carboxy terminus. This meant the carboxy terminus was the last to be synthesized. |
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In general, what are the steps to protein synthesis?
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Activation of amino acid
Initiation Elongation Termination Folding/Postranslational Processing |
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What kind of reaction activates amino acid?
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Aminoacylation
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What is the reaction for the activation of amino acids?
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1) AA + ATP --> Aminoacyl-AMP + PPi
2) Aminoacyl-AMP +tRNA --> Aminoacyl-tRNA + AMP |
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Where does the amino acid attach to the tRNA?
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3' Adenosine residue
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What kind of bond attaches amino acid to tRNA
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Ester linkage at the carboxyl end of the amino acid.
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What is aminoacylation?
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Also known as tRNA charging, it's the process of 1) activating an amino acid for protein synthesis 2) choosing the proper amino acid for a particular tRNA.
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What enzyme carriers out aminoacylation?
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Aminoacyl-tRNA Synthetase
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What distinguishes class I and II aminoacyl-tRNA Synthetase?
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Amino first transfered first or second hydroxyl of the carboxyl group.
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How many different aminoacyl-tRNA aynthetase are there?
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They are amino acid specific so there is one enzyme for every tRNA.
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How many tRNAs are there? How many codons?
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31 tRNAs for 61 codons
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In protein synthesis, what is the first step of initiation?
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30S binds to two of the initiation factors, IF-1 and IF-3
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In protein synthesis, what is needed for initiation?
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30S
IF-1,IF-3 IF-GTP 50S fMet-tRNA^(fMet) |
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In the initiation of protein synthesis, what positions mRNA onto the 30S subunit? or what selects the start site?
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3' of 16S rRNA
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In the initiation of protein synthesis, where is mRNA initially positioned?
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The AUG codon is positioned onto the P site
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In protein synthesis complex, what is the P site?
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The peptidy site because that's were the elongating peptide comes out from.
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What is the role of the IF-1 in the A site?
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To prevent any tRNA from going in prematurely
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What initiation factor binds to fMet-tRNA^(fMet)?
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IF-2-GTP
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With the exception of fMet-tRNA, where do all other tRNAs enter and bind?
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A site
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What is the final complex that's put together during initiation? What is energy requirement?
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70S initiation complex at the cost of one GTP hydrolysis
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What complicates in the formation of the initiation complex in eukaryotes vs. prokaryotes?
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Much more than three initiation factors, which is require for, among things, setting up the 5' cap binding sequence and scanning till you get to the first AUG.
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In the initiation complex of eukaryotic protein synthesis, what factor recognizes the 5' cap?
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elF4 complex
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What are the three steps of elongation in protein synthesis?
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1. Attachment of aminoacyl-tRNA to the A site
2. Peptide bond formation 3. Translocation |
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What factor binds to the incoming aminoacyl-tRNA? Why is this a proofreading mechanism?
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EF-Tu-GTP
This is proofreading mechanism because the next step cannot proceed till the GTP bound EF-Tu is hydrolyzed. |
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What happens if the aminoacyl-tRNA is incorrect?
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Aminoacyl-tRNA-EF-Tu-GTP dissociates before it can be hydrolyzed.
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In the translocation step of protein synthesis, what factor displaces the growing peptide from the A site to the P site?
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EF-G-GTP
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In the translocation step of protein synthesis, what is final location of the deacylated tRNA?
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E site
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What is the energy requirement for elongation?
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2 GTP one for binding of aminoacyl-tRNA to A site (EF-Tu) and two for translocation (EF-G)
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What binds to the stop codon?
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No tRNA, but a protein factor called RF (release factor)
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With RF at the A site, what is transferred to the
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The polypeptide chain is transferred to water.
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What factor is important for finishing termination?
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EF-G (again) and RRF replace RF
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In the termination of protein synthesis, what process promotes the dissociation of synthesis complex?
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GTP hydrolysis by EF-G
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In the termination of protein synthesis, what process helps keep the large and small subunit apart?
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IF-3
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In which rRNA subunit does the peptidyl transferase occur?
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23S rRNA
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What does EF-G tertiary structure look like?
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Aminoacyl-tRNA
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What is the energy cost per peptide bond?
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4 high energy phosphate bonds per peptide bond (aminoacylation, proofreading, translocation)
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In addition to the energy per peptide bond, what other energy cost are there?
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1 GTP for initiation per protein
1 GTP for terminaion per protein |
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What are 3 examples of post-translational modification to amino acids?
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Phosphorylation
Carboxylation Methylation |
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Why are post-translational modification so important?
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Can serve as recognition markers and regulating signaling.
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In addition to amino acid modifications, what are some other post-translational modification?
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Carbohydrate side chains
Isoprenyl groups Proteolytic processing Addition of prosthetic groups |
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In post-translation modification, where does the addition of carbohydrate side chains occur? and what's its function?
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It occurs in the ER.
It plays a key role in protein targeting. |
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In post-translation modification, what is the function of adding isoprenyl groups?
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It helps anchor otherwise soluble proteins to membranes. Regulatable and reversible.
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What does puromycin produce?
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Premature chain termination
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How does puromycin accomplish its function?
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Structurally, it resembles the aminoacyl terminus of an aminoacyl-tRNA.
So therefore, the peptide dissociates instead of translocating. |
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What are the three general locations of protein targeting?
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Membranes
Mitochondria and chloroplast Nucleus |
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What is the nature of N-terminal Signal Sequences?
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A few basic residues (Arg, Lys) followed by a few hydrophobic residues
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What does the mRNA carrying the N-terminal sequence bind to?
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SRP (signal recognition protein) which gives an arrest translation signal
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In terms of polarity, why do N-terminal sequences lead to termination?
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Since they are largely hydrophobic, they can not fold properly in the polar cytoplasm and translation needs to stop which occurs via binding of sequence to SRP.
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How is the sequence carrying the N-terminus get placed into the ER?
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The SRP is recognized by the SRP receptor in the ER
GTP hydrolysis releases the peptide from SRP and into the ER lumen Since SRP is gone, translation can proceed in the ER Signal peptidase release the peptide |
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In eukaryotes, how are proteins marked for covalent modification by ubiquitin? What does this eventually lead to?
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N-end rule which amino-terminal amino acid residues help determine the half-life of the protein
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In the process of degradating proteins marked by ubiquitin, which enzyme actually selects the condemned protein?
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Ubiquitin is attached to E1 and transfered to E2.
E3 selects the condemned protein which finally receives ubiquitin. |
