Term
| In eukaryotic cells, where are ribosomes and proteins synthesized? |
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Definition
| Ribosomes are made in the nucleolus and proteins in the cytosol |
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Term
| Why is the genetic code composed of sequences of three nucleotide bases, and not sequences of two or four nucleotides? |
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Definition
Dinucleotides (two bases) have 16 possible combinations, not enough to encode 20 different amino acids.
Tetranucleotides (4 bases) have 4 x 4 x 4 x 4 = 256 possible combinations, far too many possibilities |
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Term
| How were ribosomes identified as the site of protein synthesis? |
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Definition
| A rat was injected with radioactive amino acids, and its liver removed and homogenized. Cytosolic subcellular organelles were separated by centrifugation . Within minutes, most radioactivity was on microsomes (hot). |
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Term
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Definition
| Microsomes are mostly endoplasmic-reticulum bound ribosomes |
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Term
| After a ribosome makes protein, where are they exported? |
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Definition
| The proteins are exported into the ER lumen |
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Term
| With what was polyU incubated? |
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Definition
| It was incubated with the alkali-treated cytosolic fraction of either E coli, or liver cytosol that contained polyribosomes (microsomes; 20,000 g supernatant) |
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Term
| How did polyU sediment indicate its complementary amino acid? |
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Definition
| The sediment was radioactive only if the mixture was incubated with radioactive phenylalanine, indicating that the triplet UUU encoded phenylalanine. |
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Term
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Definition
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Term
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Definition
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Term
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Definition
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Term
| What does UGA/UGG/UAG code for? |
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Definition
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Term
| How were specific sequences of nucleotides shown to encode specific amino acids prior to elucidating the genetic code? |
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Definition
| Various trinucleotides were incubated with ribosomes purified from the alkali-treated cytosolic fraction of E. coli. Which radioactive amino acid bound to the ribosomes depended on the composition of the trinucleotide. |
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Term
| Why is the genetic code not overlapping? |
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Definition
| There would be insufficient room for aligning the aa-tRNAs next to each other to induce peptide bond formation if codons overlapped |
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Term
| Describe a filter-binding assay to identify the genetic code |
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Definition
| At high Mg+2 concentrations, ribosomal particles assemble using artificially synthesized trinucleotides and tRNA with an attached radioactive amino acid. The complex cannot pass through a filter unless the correct trinucleotide was present. |
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Term
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Definition
| A translatable mRNA sequence is an open reading frame (ORF) An ORF begins with a start codon and ends with a stop codon. |
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Term
| What is the wobble hypothesis? |
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Definition
| The idea that the third base in the codon is degenerate, allowing more than 1 codon code for an amino acid. |
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Term
1. What is the minimal number of tRNAs needed to translate all 4 codons of glycine, GGX (where X is any of the 4 nucleotide bases)?
2. How many different amino acyl transferase enzymes would be required? |
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Definition
1. 2 tRNAs, here is inadequate wobble at the third codon base to allow one tRNA anti-codon to fit all 4 codons
2. Only 1 transferase is required because all tRNAs with a given amino acid specificity are structurally indistinguishable to the tRNA amino-acylating enzyme |
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Term
| How many tRNAs are required to translate all 61 codons? |
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Definition
| A minimum of 32 tRNAs are required to translate all 61 codons (at least 31 to encode the 20 amino acids and one for initiating translation – AUG has 2 tRNAs). |
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Term
| Are prokaryotic and eukaryotic tRNAs the same? |
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Definition
| No, the tRNA utilization for each amino acid is organismspecific. |
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Term
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Definition
| Ribosomes are particles composed of structural RNA (no protein encoding function) along with many proteins. |
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Term
| How are ribosomes categorized? |
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Definition
| By size, sizes of ribosomal particles or their component RNAs are measured in Svedbergs, a sedimentation coefficient measurement |
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Term
| What are the ribosomal subunits in prokaryotes? |
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Definition
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Term
| What are the ribosomal subunits in eukaryotes? |
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Definition
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Term
| What is the S-value for a complete prokaryote ribosome? |
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Definition
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Term
| What is the S-value for a complete eukaryote ribosome? |
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Definition
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Term
| What enzyme synthesizes eukaryotic ribosomes? |
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Definition
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Term
| Where are ribosomes synthesized and processed? What does their import to the cytoplasm require? |
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Definition
| Nucleolus, import to the cytoplasm requires GTP and soluble nuclear transport factors (importins) |
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Term
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Definition
| The complex that synthesizes a polypeptide; a ribosome with mRNA and tRNA interaction |
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Term
| What are the 4 main features of a tRNA molecule? |
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Definition
1. The amino acid attachment arm
2. The dihydrouridine (DHU) arm
3. The pseudouridine arm
4. The anticodon arm |
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Term
| What is the function of the amino acid attachment arm on a tRNA? |
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Definition
| It is recognized by amino acyl synthetase and peptidyl synthetase |
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Term
| What is the function of the dihydrouridine (DHU) arm on a tRNA? |
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Definition
| Sandwiches over the pseudouridine loop when changing from cloverleaf to L-form |
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Term
| What is the function of the anticodon arm on a tRNA? |
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Definition
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Term
| What is the significance of the tRNA L form? |
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Definition
| tRNA L form spreads out the tRNA across the ribosome, connecting the complementation to mRNA on the small subunit with the peptidyl synthetase reaction on the large subunit |
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Term
| What are the unusual bases in tRNA that makes two of the "arms"? |
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Definition
| Dihydrouridine, psuedouridine |
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Term
| What does amino acyl tRNA always end in |
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Definition
CCA
The A is important because it is derived from ATP |
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Term
| How is amino acid activation for polypeptide synthesis accomplished? |
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Definition
| Activation is achieved by amino acid synthetases that bind ATP, a specific amino acid and a specific tRNA. Reaction produces pyrophosphate (PPi) AMP and aa-tRNA. |
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Term
| Why are there two classes of amino acid tRNA synthetases? |
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Definition
| Enhance synthetase specificity for the correct amino acid |
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Term
| Briefly list the two classes of amino acid tRNA and describe their difference |
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Definition
Class I: Class I tRNA synthetases transfer the amino acyl group to the 2’-OH group of ribose, releasing AMP, before moving it to the 3'OH position
Class II: Directly esterify the amino acyl group to the 3'OH position |
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Term
| What is significant of the tRNA for methionine? |
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Definition
| Methionine has 2 tRNAs: tRNAf (tRNAi in eukaryotes) and tRNAmet; one to start translation and the other to incorporate methionine into the polypeptide |
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Term
| What are the 5 main steps of protein synthesis? |
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Definition
1) Amino acid activation
2) Initiation
3) Elongation
4) Termination
5) Post-translational polypeptide processing |
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Term
| What factors does translation require? |
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Definition
3 initiation factors: IF1 IF2 & IF3
3 elongation factors: EF-Tu, EF-Ts & EF-G
3 release factors: RF1, RF2 & RF3 |
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Term
| Bacterial ribosomal assembly is initiated by two closely adjacent nucleotide sequences on the mRNA. What are they and what do they each attach? |
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Definition
Shine-Delgarno seuqnce: binds 30S
AUG sequence: binds f-met-tRNA |
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Term
| Give the order of assembly of bacterial ribosomal components to form the elongating complex. |
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Definition
1. 30S ribosomal subunit
2. mRNA
3. f-met-tRNA
4. 50S ribosome subunit |
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Term
| What are the functions of the three prokaryotic initiation factors? |
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Definition
IF1 binds to 30S, blocks a second (acceptor) site used for elongation
If2 contains GTP and binds to f-met-tRNA (50S binding hydrolyzes GTP, causing it to fall off)
IF3 binds to 30s and prevents 50S from binding until f-met-tRNA binds |
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Term
| Initiation in eukaryotes differs from that in prokaryotes in 4 major ways. What are they? |
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Definition
a) The 40S subunit binds the mRNA cap and tail.
b) The Shine-Dalgarno nucleotide sequence is absent. Instead, the 40S subunit slides along the mRNA to the AUG codon using a helicase.
c) Methionyl tRNA initiating (met-tRNAi) is not formylated. d) Greater number of initiating factors. |
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Term
| List the 4 major steps of elongation and what the respective major cofactors are |
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Definition
1) Proof-reading (EF-TS, EF-Tu, GTP)
2) Delivery (EF-TS, EF-Tu, GTP)
3) Peptide bond formation (none)
4) Translocation (EF-G and GTP) |
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Term
| How does EF-G•GDP interact with the ribosome to cause translocation? |
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Definition
EF-G• GDP stabilizes the twisted form of the ribosome. Once bound, it exchanges its GDP for GTP.
Hydrolysis of the GTP to GDP translocates the ribosome to the post-translocation state |
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Term
| What is unique about the codon for transcription termination? |
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Definition
| Signaled by termination codons that bind to a release factor (a protein) instead of an amino acyl tRNA |
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Term
| What is the difference between the functions of release factor (RF) and ribosomal recycling factor(RRF)? |
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Definition
RF releases the polypeptide and allows EF-G●GTP to attach to the empty P site
RRF attaches to EF-G●GTP and is released along with it and the large subunit after hydrolysis of the EF-G●GTP |
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Term
| How many ATP equivalents are needed to synthesize a polypeptide during translation? |
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Definition
4 ATP equivalents per amino acid
(2 ATP to activate aa-tRNA,
1 GTP for delivery,
1 GTP for translocation) |
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Term
| What are the 6 functions of the ribosomes during protein synthesis? |
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Definition
1) Initiates translation by 30S subunit binding to Shine-Dalgarno sequence or eukaryotic mRNA cap
2) Channel for mRNA to interact with aa-tRNA
3) GTPase on its 50S subunit to ensure initiation, delivery, translocation and release
4) GDP/GTP exchange factor for translocation and release
5) Peptidyl transferase (a ribozyme) in the large subunit
6) Tunnel in the 50S subunit for the peptide to escape |
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Term
| Which of the following is not an antibiotic: Erythromycin (an aminoglycoside), Tetracycline (or Doxycycline), Cycloheximide, Puromycin, Streptomycin, Chloramphenicol? |
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Definition
| Cycloheximide. It binds only to a eukaryotic ribosome. Unlike all the others, cycloheximide cannot inhibit bacterial protein synthesis. It does not affect bacterial growth and is therefore not an antibiotic |
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Term
| Describe the function of puromycin |
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Definition
Anti-cancer drug
Acts as a releasing factor, producing toxic incomplete peptides |
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Term
| Describe the function of cycloheximide |
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Definition
Anti-cancer drug
Stops protein synthesis by binding to 60S and inhibiting translocation No effect on prokaryotes |
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Term
| Describe the function of erythromycin |
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Definition
Antibiotic
Binds to 50S subunit and inhibits translocation |
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Term
| Describe the function of chloramphenicol |
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Definition
Antibiotic
Binds to 50S subunit where it inhibits peptidyl synthetase in 50S |
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Term
| Describe the function of tetracycline |
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Definition
Antibiotic
Inhibits delivery by binding to 50S |
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Term
| Describe the function of streptomycin and aminoglycosides |
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Definition
Antibiotic
Inhibits initiation and proofreading by binding to 30S Leads to antibiotic resistance |
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