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75 Cards in this Set
- Front
- Back
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A ribosome can be considered a....
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ribozyme. The RNA components play the most fundamental parts.
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The link between A.A. and tRNA is made by a
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tRNA synthetase
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What is the average speed of translation?
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20 amino acids per second
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For translation to provide an adequate protein, the error rate must not exceed...this number to produce proteins with as many as 1000 amino acids
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10 to the negative 4
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How many nucleotides long are tRNA's?
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73-93
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3' CCA - OH
what is this? |
amino acid attachment site
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All tRNA's have a common structure. What are the features?
(6) |
1. Single chain of 73- 93 ribonucleotides
2. Contain unusual bases. Modified. Methylated, dimethylated...that stuff. 3. Half of the nucleotides are base paired, other half makes up 3' CCA attachment site, t psi C loop, extra arm, anticodon loop, DHU loop. 4. Phosphorylated 5' end. Usually pG. 5. The amino acid is attached to a hydroxyl group at the 3' CCA end. 6. The anticodon is present in a loop near the center of the sequence. |
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tRNA structure continued
1. What shape is a tRNA molecule? 2. Four helical regions arrange to form how many continuous segments of double helix? 3. What type of DNA are these segments similar to? 4. Do bases in non helical regions participate in hydrogen bonding? 5. The 3' CCA acceptor stem is double or single stranded? |
1. L shaped
2. 2 3. A-DNA 4. Yes, although they may not be watson-crick base pairing 5. single stranded, allowing it to change conformation for amino acid activation and protein synthesis |
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Linkage of amino acid to tRNA is crucial for two reasons:
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1. attachment of tRNA to AA establishes genetic code
2. Peptide bond formation between free amino acids is not energetically favorable. So, intermediate is formed. Names of this intermediate: Aminoacyl tRNA Amino acid esters Charged tRNA |
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Charging tRNA: 1st step
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Producing ester linkage by adenylation of amino acid.
A.A. + ATP --> aminoacyl-AMP + PPi |
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Charging tRNA: second step
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transferring aminoacyl portion of aminoacyl-AMP to tRNA to form aminoacyl-tRNA
Aminoacyl-AMP + tRNA --> aminoacyl-tRNA + AMP + PPi |
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How many equivalents of ATP is consumed in the synthesis of each aminoacyl tRNA?
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two.
One in forming the ester linkage of aminoacyl-tRNA, and the other is consumed in driving the reaction forward |
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Aminoacyl-AMP can also be called
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Aminoacyl adenylate
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During the charging reaction, does the Aminoacyl tRNA synthetase disassociate from the intermediate?
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No.
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What special adaptation does theronyl-tRNA synthetase have for selectivity?
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A zinc ion.
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tRNA synthetase mediates proofreading with...
1. what kind of site? 2. what kind of chemical reaction? 3. rejects the A.A.'s that are too big with which site? 4. cuts the A.A.'s that are too small with which site? 5. term for two checks? |
1. editing site
2. hydrolysis 3. activation site 4. editing 5. double sieve |
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Proofreading has been selected in evolution only when
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initial binding is not accurate enough, and fidelity must be enhanced.
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If anticodon loop is completely lacking from tRNA, can the tRNA synthetase still recognize the tRNA?
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In some cases, yes. A microhelix may provide enough evidence. But it usually recognizes via anticodon loop. Attaches to both loop and acceptor stem.
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Class I synthetase
1. CCA arm conformation 2. acylate which hydroxyl group of tRNA? 3. how many units? Class II synthetase 1. CCA arm conformation 2. Acylate which hydroxyl group? 3. How many units? Overall: Two classes bind to same face or different faces of tRNA? Two classes bind ATP in same or different conformations? One of two classes specific for how many A.A.'s? |
1. Helical
2. 2' OH group 3. monomeric 1. Hairpin conformation 2. 3' OH group 3. Dimeric Different faces Different conformations 10 |
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Activation and transfer steps are both catalyzed by the same aminoacyl tRNA synthetase.
True or False |
True
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Aminoacyl AMP tightly bound to synthetase by what kind of interactions?
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Non covalent interactions. It's relatively stable in the absence of tRNA.
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Serine may be mistakenly put on a threonine aminoacyl tRNA synthetase base it's hydroxyl group. What does this increase the error rate to?
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10-2 to 10-3
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Ribosome composition
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70s ribosome unit, made of 30s and 50s units
30s unit 21 proteins 16s rna 50s unit 34 proteins 23s rna 5s rna |
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Can the 30s and 50s ribosome subunits each be reconstituted from their rna and protein parts, in vitro?
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Yes. supramolecular complexes can form spontaneously from their macromolecular constituents.
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what does the interface between 30s and 50s subunits of a ribosome consist of?
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RNA entirely.
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How is secondary structure of rRNA discovered?
How is tertiary structure discovered? |
1. Sequence comparison, chemical studies.
2. X ray crystallography. |
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rRNAs are folded into defined structures with many
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short duplex regions
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Which plays a more active role in the ribosome, the rRNA, or the proteins?
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the rRNA. key sites in the ribosome are composed of nearly entirely rRNA.
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What is the direction of translation?
why is this important for prokaryotes? |
5 to 3
Transcription and translation can occur at the same time. Forms polyribosomes, or polysomes |
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True or False:
All known mRNA molecules contain sequences that signal the beginning and end of each polypeptide chain. |
True
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50% of mRNA sequences begin translation with Pwhat A.A?
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Met
(AUG) others starts with GuG (valine) and even less with UUG (leucine) |
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How, experimentally, did they find mRNA start sequences?
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Used pancreatic ribonuclease to digest mRNA ribosome complexes involved with initation.30 nuceleotides were protected from digestion, because the ribosome was covering them during initiation.
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What is the shine delgarno sequence?
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Purine rich area upstream of AUG start codon that attaches to the 3' end of the 16s rRNA.
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What are two interactions that determine where protein synthesis starts?
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1. pairing of mRNA bases (shine delgarno) with 3' end of 16s rRNA
2. pairing of initiator codon AUG with anticodon of a tRNA initiator molecule |
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True or false:
The same tRNA synthetase is used to charge fMet-tRNA and Met-tRNA |
true
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True or false:
Free methionine and methionyl-tRNAm are substrates for the transformylase |
false
it uses met-tRNAf |
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Where is the only spot on a polypeptide chain you will find f-met?
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the n terminus
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Formylation of Methionyl-tRNA:
1. what part of the methionyl group is formylated? 2. What is the formyl donor? 3. What is the enzyme? 4. What is the product? |
1. amino group
2. N10-formyltetrahydrofolate 3. transformylase 4. formylmethionyl-tRNAf (fMet-tRNAf) + tetrahydrofolate |
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Which esters work? 2' or 3' linkages between tRNA and the AA?
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only 3'. if it's 2', then transesterification to the 3' ester
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Where does the growing peptide chain go while being synthesized?
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it escapes through the tunnel somewhere near the P site
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What subunit does the mRNA go through?
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the 30s subunit
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What catalyzes the transfer of the amino acid in the P site to the amino group of the amino acid in the A site?
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peptidyl transferase center, located at the base of 23s rRNA (of large subunit)
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How is mRNA moved along the ribosome?
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With the help of Elongation Factor G, powered by the cleavage of GTP
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If a tRNA is mischarged, will it still attach its incorrect A.A. to the corresponding codon?
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Yes. tRNA's attached amino acid plays no part in codon selection.
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Allowed pairings at the wobble position: 1st base of anticodon, 3rd base of codon
1. C 2. A 3. U 4. G 5. I caugi |
1st anticodon 3rd codon
C G A U U A G G U C I A C U |
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True or false: Codons that differ in either of their first two bases must be attended by different tRNAs.
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True
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What base allows the greatest wobble and how is it formed?
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Inosine, formed by deamination of adenosine
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Why is wobble tolerated in the third position but not in the first two?
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The 16s rRNA has 3 highly conserved bases that check the base pairing of the first and second codons by hydrogen bonding with them.
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Does the ribosome play an active role in decoding codon anticodon interactions?
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Yes, with the checks made by the highly conserved bases on the 16s rRNA
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Translation initiation
1. IF1 2. IF2 3. IF3 |
1. binds close to A site to tRNAf will bind to P site
2. G protein that binds to GTP, then to tRNAf, then to mRNA 3. binds to 30s subunit to prevent premature binding to 50s that would form a 70s devoid of mRNA or tRNAf |
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Translation elongation factors
1. EF - Tu 2. EF - Ts 3. EF - G |
1. G protein that binds to tRNA's and directs them to their correct codon on the mRNA. Used GTP. Binding protects ester linkage, and increases fidelity.
2. joins the EF-tu complex, releases GDP. Then Ef-tu is free to go on 3. G protein that binds to 23s unit of 50s rRNA. Hydrolysis of GTP causes tRNA in A site to go to P site. mRNA and deacylated tRNA go with it |
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Release Factors
1. RF1 2. RF2 3. RF3 |
1. UAA and UAG
2. UAA and UGA 3. mediates rf1 and rf2 binding |
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Eukaryotic translation: how its different than prokaryotes
1. initiation 2. initiation tRNA 3. structure of mRNA 4. Ribosome 5. Elongation and termination |
1. AUG always start codon. Always one initiation site. tRNAi + 40s ribosome unit start at methyl cap and scan until AUG is found. No shine delgarno sequence.
2. Met-tRNAi 3. mRNA is circular. eIF-4e and eIF4g link poly a tail to methyl cap head 4. 40s and 60s make 80s 40s 18s rRNA 60s 5s 28s 5.8s 5. termination is carried out by single release factor |
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What are the three classes of proteins made by the rER?
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Secretory proteins
Lysosomal proteins Proteins spanning plasma membrane |
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SRP targeting cycle
4 things |
1. Signal sequence - free ribosomes produce 9 to 12 hydrophobic A.A. residues
2. SRP - consists of 7s RNA and 6 proteins. Binds tightly to ribosomes that emit the signal. 3. SRP receptor - docking protein. 4. Translocon - made of peripheral and integral proteins that bind ribosomes and project growing chain into lumen. notes - SRP and SR simultaneously hydrolyze bound GTPs...they are gtpases |
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Transport of protein after injection into lumen
1. cargo receptor 2. Coat Proteins 3. V-snare 4. T-snare |
1. integral membrane protein, binds cargo by recognizing AA sequence or added carbohydrate.
2. Facilitates formation of buds. Proteins pinch off vesicle. 3. Coat protein is shed, reveals this, another integral protein. binds to a particular t-snare 4. what the vesicle's v snare binds to. located on target membrane. |
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Puromycin
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Causes premature chain termination by acting as an analog of aminoacyl tRNA. Strengthened by alpha amino group
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Streptomycin and other aminoglycosides
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inhibits initiation by messing with fmet-tRNA-f
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Diphtheria toxin
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Block EF2, halting translocation of growing polypeptide chain
involves dipthamide, a modified histidine residue |
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Ricin
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acts on adenine substrate of ribosome, preventing EF attachment
a chain - does the dirty work b chain - binds to the cell membrane |
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Constituted expression
Regulated expression |
when all genes are expressed all the time
when genes are expressed only at certain times |
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How is gene expression controlled mostly? at what level?
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The level of transcription
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What is a recurring theme in protein dna interactions?
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symmetry matching
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How is the lac operon repressed?
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by a repressor protein, a dimer. It fits a double helix nicely into the major groove of the dna, and is symmettric like the dna itself.
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What is the most commonly seen dna binding unit in prokaryotes?
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helix turn helix motif
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E. coli methionine repressor:
what is the structure? |
B strands
inserted into major grooves |
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Basic Leucine zippers
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basic region attaches to and recognizes dna, inserts into major groove
leucine zipper region is a coil of coils that contain many leucines. stabilizes the protein heterodimer. |
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Zinc Finger Domain
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Domain made of 2 cysteine and 2 histidine residues that bond to a zinc atom. forms a string that follows major groove of dna. can cover large stretches. An alpha helix makes contact within the groove
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What are the genetic elements of the lac operon
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The regulator gene
the operator site set of structural genes |
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Lac repressor structure
1. what kind of motif 2. what area binds to DNA |
1.helix turn helix
2. amino terminal |
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What is the inducer of the lac operon?
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Allolactose
but, can also be induced in the lab by IPTG |
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Induction in the lac operon
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Allolactose or IPTG binds to both monomers of regulatory unit. Unit disassociates from DNA. lac mRNA resumes.
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Pur repressor is also known as a
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corepressor. represses only when bound to a small molecule. this is different than the lac repressor, which represses when NOT bound to a small molecule
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Catabolite repression
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High levels of glucose lower level of cAMP. Don't need other enzymes for digestion, just glucose.
When cAMP is high, it binds to the dimer CAP. cAMP-CAP complex binds to position at -61 with inverted repeat. adjacent to where rna polymerase binds. increases initiation by a factor of 50 |
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trp operon attenuation
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attenuation is when transcription is broken off before any good mRNA is produced.
leader sequence encodes 14 AA including tryptophan, and attenuator region that form hair pin loop if tryptophan is present. |
