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130 Cards in this Set
- Front
- Back
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What occurs in translation?
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The cell interprets the mRNA's code to make proteins.
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What is the basic scheme of translation?
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mRNA acts as a template for interaction of ribosomes and tRNA.
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What enzyme is responsible for attaching amino acids to tRNA?
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Aminoacyl synthetase
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What direction (in terms of chemical bonds) are proteins made?
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Amino to carboxyl direction.
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Where in the cell does protein translation occur?
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In the cytosol.
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List the 7 components of Protein Synthesis:
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1. Amino acids
2. tRNAs 3. mRNAs 4. aa-tRNA synthetases 5. Ribosomes 6. Initiation, elongation & termination factors 7. ATP/GPT energy sources |
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How many tRNAs are actually in humans?
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>50
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What are the 2 important sites on a tRNA?
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1. AA Attachment site (3' end)
2. Anticodon sequence |
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In E.coli, where does regulation of translation take place primarily?
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At the level of TRANSCRIPTION
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What are aa-tRNA synthetases?
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A family of enzymes that attach AA's to their tRNAs in a very specific 2-step rxn.
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What are ribosomes?
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Ribonucleoprotein complexes consisting of TWO subunits.
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What ribosome is for
-Prokaryotes? -Eukaryotes? |
Prok: 70S
Euk: 80S |
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What subunits make up the ribosomes in
-Prokaryotes? -Eukaryotes? |
Prok: 30S + 50S
Euk: 60S + 40S |
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Where in euks are ribosomes found (3 places)?
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-Free
-RER - rough endopl. reticulum -mitochondrial |
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What is RER for?
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Rough endoplasmic reticulum synthesizes exported and membrane-bound proteins.
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How much energy is required for the addition of one amino acid?
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4 ATP and GTP; more for added elongation and termination.
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How is energy obtained for elongation?
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Via the cleavage of high energy bonds of ATP/GTP.
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What makes up the 50S subunit of proks?
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rRNA: 23S + 5S
Proteins: L1/L2/L3 (31 total) |
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What makes up the 30S subunit of proks?
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rRNA: 16S
Proteins: S1/S2/S3 (21 total) |
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What does binding of the 50S and 30S subunits make?
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The 70S Assembled ribosome!
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What makes up the 60S subunit of euks?
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rRNA: 28S + 5.8S
Proteins: L1/L2/L3 (50 total) |
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What makes up the 40S subunit of euks?
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rRNA: 18S
Proteins: S1/S2/S3 (33 total) |
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What are codons?
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Triplet sequences that make up the genetic code.
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How many different combinations of bases are there in the genetic code?
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64 -> 4^3
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How many of the possible codons code for amino acids?
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61
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What do the other 3 do?
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Act as nonsense/termination codons.
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How many amino acids are there?
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20.
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So what does it mean that there are 61 codons for 20 amino acids?
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The genetic code is redundant - more than one codon exists for each amino acid.
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List the 4 properties of the Genetic Code:
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1. Specific - codons always encode same amino acid.
2. Universal 3. Redundant 4. Non-overlapping and commaless. |
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What are the 3 termination codons?
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-UAG
-UGA -UAA |
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What are 3 types of alterations that can occur in codons?
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1. Silent mutations
2. Missense mutations 3. Nonsense mutations |
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What are silent mutations and their result?
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Change in codon base to a different base that results in a different codon - BUT it encodes the same amino acid.
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What are missense mutations and their result?
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Replacement of a codon base that results in a DIFFERENT amino acid being encoded.
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What are nonsense mutations and their result?
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Base replacement that results in a Termination codon being encoded. (UAA/UGA/UAG)
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How do tRNA molecules read mRNA?
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Anticodons are 'flipped' so they read mRNA 5'->3'
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So which base sequence dictates the amino acid? mRNA or the anticodon?
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mRNA!!! That's where the codon is. If you read the anticodon, it's backward.
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Where is the wobble position?
-In the Anticodon -In the Codon |
Anticodon: 5' position
Codon: 3' position |
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How many codons can be recognized if the wobble position is occupied by
-Cytosine -Adenine |
1
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How many codons can be recognized if the wobble position is occupied by
-Uracil -Guanine |
2
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What codons can be recognized when wobble is occupied by
-Uracil? -Guanine? |
Uracil: Adenine or Guanine
Guanine: Cytosine or Uracil |
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What residue confers capability of recognizing 3 codons when in wobble pos?
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Inosine
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What bases can be recognized by inosine?
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Adenine
Uracil Cytosine |
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There are 61 anticodons for 20 amino acids; do there have to be 61 tRNA's then?
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No; by using the wobble position, fewer tRNAs have to exist in order to READ all 61 codons, and still polymerize 20 amino acids.
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Nutshell: what is the use of the Wobble hypothesis?
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There need not be 61 tRNAs to read all 61 codons.
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What is a nonsense suppressor tRNA?
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A tRNA that has mutated
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When looking at mRNA to see how it's read, which direction directs codon?
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5'->3'
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What are Suppressor tRNA's?
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TransferRNA molecules that have mutated, so that their anticodons recognize STOP (nonsense)codons, but yet they transfer an amino acid to the growing chain.
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What is the result of suppressor tRNA in the overall translation scheme?
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Normal suppression (stopping) does not occur; the chain keeps on growing instead.
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What are the 5 steps in protein synthesis?
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1. Activation of Amino Acids
2. Initiation 3. Elongation 4. Termination/Release 5. Folding/Processing |
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What are the 5 essential components of Amino Acid Activation?
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1. 20 Amino Acids
2. 20 Aminoacyl-tRNA synthetases 3. 20 or more tRNAs 4. ATP 5. Mg2+ |
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What are the 8 essential components of Initiation (step 2)?
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1. mRNA
2. N-formylmethionyl-tRNA 3. Start Codon in mRNA (AUG) 4. 50S ribosome subunit 5. 30S ribosome subunit 6. Initiation Factors 1/2/3 7. GTP 8. Mg2+ |
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List the 5 essential components of Elongation:
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1. Functional 70S ribosome
2. AA-tRNAs specific to codons 3. Elongation Factors Te/Ts/G 4. GTP 5. Mg2+ |
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What are the 3 essential components of Termination and Release (step 4)?
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1. Termination codon in mRNA
2. Polypeptide release factors 3. ATP |
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What are the polypeptide release factors called?
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RF1
RF2 RF3 how fitting! |
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How do AA-tRNA synthetases are so specific and accurate?
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They recognize not only the Anticodon arm, the AA attachment point, and also motifs on other arms of the tRNA
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Why do amino acids have to be charged prior to attachment to their tRNA?
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Because they are not naturally in an energy state that favors the transfer.
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What provides the energy for amino acid activation?
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PPi hydrolysis
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What is the actual reaction that takes place in activating amino acids?
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Nucleophilic attack of the alpha phosphate by the terminal O of the carboxyl group on the amino acid; subsequent release of PPi.
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How are activated amino acids put onto their tRNAs?
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The AMP is a good leaving group; the 2' or 3' -OH on the tRNA's adenine residue attacks the carbonyl group on the AA with release of AMP.
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What's the difference between Class I and Class II AA-tRNA synthetases?
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Class I uses the 2' OH
Class II uses the 3' OH |
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What's the primary difference to remember between Prok and Euk mRNAs?
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Prok = polycistronic
Euk = monocistronic |
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How many rRNA molecules are contained in:
-Prokaryotic ribosomes -Eukaryotic ribosomes |
Prok: 3 - 23S, 5S, and 16S
Euk: 4 - 28S, 5S, 5.8S, & 18S |
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What are the APE sites on ribosomes?
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The binding sites for tRNA
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In bacteria, how do ribosomes know which AUG encoding MET is really the start point?
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By recognizing the Shine Dalgarno sequence
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What is the Shine Dalgarno sequence?
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Sequence 6-10 nucleotides upstream the REAL start codon; purine rich (UAAGGAGG)
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What component of the ribosome recognizes the Shine Dalgarno sequence?
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The 3' end of the 16S ribosome (on 30S subunit);
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Where is the Shine Dalgarno sequence located?
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Near the 5' end of the prokaryotic mRNA; upstream the first start codon (aug)
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How do eukaryotic ribosomes know when to start translating the mRNA?
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There's only one start because their genes are monocistronic.
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What signals eukaryotic ribosomes to assemble for translation?
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The 40S ribosomal subunit binds and moves down the mRNA until it encounters AUG.
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Why is it important that prokaryotic 16S ribosomes bind the Shine Dalgarno sequence?
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So that the start codon is put into the P site, not the A site.
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What is always the first amino acid in protein?
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Methionine
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What special tRNA puts the methionine into the P site in bacteria?
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Initiator tRNA
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What is special about the initiator tRNA?
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It carries N-formyl methionine
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Why do prokaryotes need the first AA to be N-formyl methionine?
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Because the N is blocked from making a peptide bond and so this can only be placed at initiator P sites.
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What enzyme is responsible for formylating the methionine?
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Transformylase
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what's important to remember about how transformylase works?
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It only recognizes fmet-tRNA the special initiator tRNA for protein synth in proks.
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What is different about translation inititation in eukaryotes?
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There is no shine-dalgarno sequence and no formylated methionine; just normal MET.
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What else aids in initiation?
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INITIATION FACTORS
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Which IF helps recognition of the initiation codon in both euks and proks?
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Euks: eIF-2
Proks: IF-2 |
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What does IF3 do?
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-Promotes dissociation of 50S and 30S ribosome subunits
-Aids in 30S binding mRNA |
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What does IF1 do?
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Helps IF3 by increasing the dissociation rate of the subunits.
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What does IF2 do?
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Complexed w/ GTP; Binds fmet-tRNA and puts it in the P site.
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How do the IF molecules leave when initiation is done?
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IF3 leaves and makes GTP hydrolyze to GDP. That makes IF2-GDP release; the hydrolysis also helps IF1 release, and the 50S unit can now associate with 30S.
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What antibiotic interferes with protein initiation and how?
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Streptomycin; binds the 30S subunit, distorting it so it can't bind mRNA.
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What are the 3 steps in Translation Elongation?
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1. 2nd Aminocyl-tRNA binds at the A site.
2. Peptide bond formation between the 2 amino acids. 3. Release of uncharged AA and Translocation. |
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What aids in the first step of elongation?
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EF-Tu-GTP; GTP hydrolyzes when the AA-tRNA binds A site.
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What happens to EF-Tu-GDP?
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EF-Ts binds to make it release GDP; then GTP binds and causes EF-TS to release. Generates a fresh EF-Tu-GTP
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How does EF-Tu actually help protein synthesis?
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By a mechanism called kinetic proofreading.
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What is kinetic proofreading?
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When AA-tRNA binds the A site, the anticodon must bp with mRNA; otherwise it will leave the A site before GTP hydrolysis can occur.
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What forms the peptide bond in the 2nd step of elongatn?
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Attack of the already-present aa's ester bond between tRNA and AA by the amino group on the incoming aa.
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What catalyzes transpeptidation?
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Probably the 23S ribozyme peptidyl transferase
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What else happens in step 2, transpeptidation?
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The 3' and 5' ends of the uncharged tRNA bend into the E site, and the growing amino acid chain bends into the P site; their anticodons remain in the A and P sites though.
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What happens in translocation?
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Dissociation of the uncharged tRNA; Then, complete shift of the ribosome on the mRNA so that the A site is empty and P/E are now occupied.
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What allows for translocation to take place?
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GTP hydrolysis on EF-G
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Which steps of elongation require GTP?
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steps 1 and 3.
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What signals termination of protein synthesis to occur?
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The presence of a termination codon on the mRNA in the A site.
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What happens when a termination codon is encountered in the A site?
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A release factor RF binds the A site, breaking the ester bond of the peptide chain and the tRNA in the P site.
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What is required for termination?
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ATP (as well as termination codon and release factors)
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What is protein targeting?
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Incorporation of specific sequences encoding signals in proteins, to help them get to their functional destination after cytosolic production.
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What does it mean to say protein elongation is fast and accurate?
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Fast: 15-20 AA added / sec
Accurate: ~1 mistake for every 10000 AA added. |
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What is EIF4?
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Eukaryotic Initiation Factor 4
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What does EIF4 do?
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Guides mRNA to bind the pre-Initiation complex for translation by recognizing the 5' Cap on mRNA.
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What happens after the mRNA binds to the small subunit?
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The initiation complex scans the mRNA for AUG; when it finds it, the Met-tRNA is positioned and eIF5 helps the 60S subunit bind 40S.
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Why is EIF4 so gosh darn important?
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It's a key step in regulating the translatability of mRNA; if there's no eIF4, the pre-initiation complex won't recognize the cap
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What is required for the scanning-for-AUG action during eukaryotic initiation?
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Helicases and Energy (ATP)
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What feature re: eIF4 allows us to manipulate its effectiveness for recognizing mRNA's cap?
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the fact that it must be phosphorylated to be active.
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By manipulating eIF4 through phosphorylation, what power does that give us?
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The ability to regulate the translatability of a eukaryotic cell's RNA.
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HOW do we manipulate eIF4?
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Phosphorylate: activates eIF4, makes it recognize cap, guide mRNA to 30S ribosome. Translation then ensues.
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Name 8 factors that stimulate translation by phosphorylating eIF4:
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1. TNF-alpha
2. Insulin 3. Epidermal growth factor 4. Plt-derived growth factor 5. Nerve growth factor 6. IL-1 7. pp5 src 8. p21 ras |
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Name one thing that inhibits eIF4 (cap binding protein):
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Polio virus
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How does polio virus inhibit eIF4?
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By proteolysing it; this allows the virus' own uncapped RNA to be translated.
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When is eIF4 not phosphorylated?
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In the eggs of urchins and frogs, and in Heat Shock.
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How does Diphtheria toxin work?
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It inhibits translocation during eukaryotic protein translation.
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How does the diphtheria toxin inhibit translocation?
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It ribosylates EF-2 translocase, which prevents elongation.
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What is EF-2?
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The eukaryotic analog to EF-G in proks, which translocates the ribosomal machinery.
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Why is the diphtheria toxin so potent?
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It is catalytic; it inactivates ALL EF-2 molecules in the host cell.
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What molecule does the diphtheria toxin take advantage of?
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Diphthamide; a modified His residue within the EF-2 molecule.
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How is Diphthamide made?
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It's essentially a His residue, but with ADP (minus a nicotinamide) on it.
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So name 3 antibiotics that inhibit prokaryotic translation.
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-Streptomycin
-Tetracyclin -Chloramphenicol |
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Name an antibiotic that inhibits eukaryotic translation.
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Cyclohexamide
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What type of an antibiotic is streptomycin?
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An aminoglycoside.
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How do aminoglycosides and streptomycin work?
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By inhibiting translation INITIATION, and misreading of mRNA.
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How does Tetracycline work?
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Binds the 30S subunit and inhibits binding of aa-tRNAs.
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How does Chloramphenicol work?
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Inhibits peptidyl transferase activity of 50S subunit in prokaryotes..
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How does Cyclohexamide work?
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Inhibits the peptidyl transferase activity of 60S subunit in eukaryotes.
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How does Erythromycin work?
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Binds the 50S subunit and inhibits translocation.
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How does Puromycin work?
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Acts as an analog of aa-tRNAs in proks/euks, causes premature chain termination.
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Why does Puromycin terminate translation?
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It results in an amide linkage between the puromycin molecule and the AA in the P sites; this disrupts the peptide bond formation.
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How do bacteria become resistant to erythromycin?
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They contain a plasmid-born methylase, which converts a single adenosine in 23S rRNA to N6-dimethyl adenosine, which blocks the binding site of the drug.
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What are 6 types of post-translational modification?
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1. Proteolytic processing
2. Phosphorylation 3. Glycosylation 4. Hydroxylation 5. Additional modifications 6. Ubiquitination |
