translation Flashcards
are amino acids coded by codons or anticodons
codons
what are codons
3-nucleotide codes for amino acids
is the codon on the mRNA or the tRNA
mRNA
is the anticodon on the mRNA or the tRNA
tRNA
function of tRNA?
required to convert from nucleotide to amino acid
where does translation occur in the cell
cytosol
where in the cytosol does translation occur
on ribosomes
which main enzyme was important for researchers to be able to determine the nucleotide code
polynucleotide phosphorylase
describe the actions of polynucleotide phosphorylase
in the presence of NDPs it will create a polynucleotide without a template
describe how UUU was the first codon to be assigned (ie how they figured out UUU=Phe)
they incubated polyU mRNA with E coli extracts and one radioactive amino acid (repeated for all 20 amino acids). Only one would produce a radioactive protein, and this would be the one that codes for UUU
describe how researchers determined the code for codons with two different nucleotides (As and Cs)
used 5 parts ADP and 1 part CDP + polynucleotide phosphorylase. They determined the likelihood of each possible codon based on the amounts that were added. Based upon the protein product, they determined which aa’s were produced in which amounts
the first base of a codon pairs with the ___ base of the anticodon
third
T or F: codon and anticodon are antiparallel
true
some tRNAs have which weird nucleotide
inosinate (I)
which position of the anticodon is inosinate found in
the third position of the anticodon
which nucleotides of the codon can inosinate base pair with
A U and C
T or F: base pairs of codon-anticodon that contain inosinate are stronger than watson-crick pairings
false; they’re weaker
when more than one codon specifies an amino acid, which nucleotide is often the different one
the third nucleotide of the codon
what is the wobble hypothesis
first two bases of the mRNA codon are under strong watson-crick pairings with the anticodon and confer most of the coding specificity. The third base only pairs loosely, allowing the rapid dissociation of the tRNA during synthesis. Reduces the need for accuracy and increases speed. These rules allow for only 32 tRNA molecules to translate all 61 codons
how many tRNA molecules are needed to translate all 61 codons
32
describe how the wobble hypothesis helps maintain genetic integrity
if there is a random mismatch, there is a 1/3 chance it will be in the wobble position, and therefore may still code for the same amino acid (= silent mutation)
what is the most common type of mutation
transition mutation
what is a transition mutation
a purine is replaced with another purine, or a pyrimidine with another pyrimidine
describe why transition mutations aren’t THAT bad
mismatches in codon positions 1 or 2 tend to change the amino acid, but introduce a similar type of amino acid (ie the product is still hydrophobic, so the damage isn’t that bad)
T or F: some mRNAs are edited before translation
true
in which RNAs does RNA editing often occur
in RNAs made from genes in the mitochondria and chlorplast
describe what happens when RNA editing involves addition or deletion of bases
shifts the reading frame
shifting the reading frame requires which molecule
guide RNA (gRNA)
describe the actions of gRNA
it will pair to the mRNA at the editing site and direct several proteins (endo/exonucleases, ligases, etc) to it. These proteins will introduce extra bases
which weird type of pairing can occur during frame-shift mutations
G-U
in deamination RNA editing, describe what happens to C and A bases
C becomes U
A becomes I
what are Alu elements
repetitive sequences that often exist in introns + the untranslated regions at the end of transcript
in which part of the genome do deaminations occur
Alu elements
describe how RNA editing leads to different apoB proteins in different tissues
apoB-100 in synthesized in the liver for LDLs and apoB-48 is synthesized in the intestine for chylomicrons. Both are encoded for by the same mRNA which will produce the liver version without editing. However, a cytidine deaminase found only in the intestine can bind to the mRNA at CAA –> UAA. UAA is a stop codon. The shorter apoB-48 is thus translated in the intestine only
bacteria ribosomes are __% rRNA and __% protein
65 and 35
how many nm long are ribosomes
18
T or F: in bacterial ribosomes, there are no proteins near the active site
true
how many rRNAs are in the 30S subunit
1
how many rRNAs are in the 50S subunit
2
describe the structure of the ribosome in regards to rRNA and protein
rRNA have extensive intrachain base pairing and form the structural core of the complex. The proteins have domains on the ribosome surface, but also extensions that protrude into the rRNA core for structural stability
how many rRNAs are in the 40S subunit
1
how many rRNAs are in the 60S subunit
3
how many amino acids long is tRNA
73-93
which nucleotide is at the 5’ end of tRNA
G
which 3 nucleotides are at the 3’ end of tRNA
CCA
how many unpaired bases are on the anticodon arm of tRNA
7
what is a polysome
a big string of ribosomes all in a row, translating the mRNA as it has just been synthesized
which enzymes place the appropriate amino acid on the amino arm of the tRNA
aminoacyl-tRNA synthetases
describe the linkage that binds the aminoacyl group to a tRNA
the aminoacyl group is esterified to the 3’ OH on the terminal A residue of the tRNA
what stage must occur before translation initiation
activation of amino acids
how many aminoacyl-tRNA synthetases are there
20; 1 per amino acid
what does it mean for an amino acid to be activated
amino acid + tRNA + ATP –> aminoacyl-tRNA
how many ATP are required to make one aminoacyl-tRNA
2 ATP
describe the 2 step formation of aminoacyl-tRNAs
formation of an intermediate occurs first: aminoacyl adenylate (aa + AMP). The enzyme then binds to this intermediate. aminoacyl group is transferred from the enzyme to the corresponding tRNA
why is it helpful to have 2 steps to produce aminoacyl-tRNA
each step is a chance to notice any errors before we continue
T or F: ribosomes can proofread their transcript
false; they cannot proofread
what are the implications of ribosomes being unable to proofread
if the wrong aa gets added to the tRNA by the synthetase, there’s no way to catch the mistake later
which aa does the start codon AUG code for
methionine
describe how the initiation methionine is different from internal methionines
it’s attached to a formyl group = fMet
T or F: fMet is found in both prokaryotes and eukaryotes
false; it’s only in prokaryotes
T or F: fMet and Met have different tRNAs
true
pro initiation: name the consensus sequence important for initiation
Shine-Dalgarno sequence
describe the structure, location, and binding partner of the shine-dalgarno sequence
- 4-9 purines
- found 10 nucleotides upstream of AUG
- binds to pyrimidine rich 16S rRNA in the 30S subunit
pro initiation: benefit of the SD sequence?
positions AUG over the P site of the ribosome. It does this by pairing to the 16S rRNA in the 30S subunit
name the 3 ribosome sites
aminoacyl site, peptidyl site, exit site
list what each ribosome site can bind
A and P bind aminoacyl-tRNAs, E can only bind uncharged tRNAs
describe which subunits of the ribosome form the A P and E sites
A and P are formed by both large and small, but E is mainly on the large
pro initiation: role of IF1
blocks tRNA-fMet from entering the A site
pro initiation: role of IF3
prevents premature binding of 50S subunit
pro initiation: what occurs after IF1 and IF3 do their jobs
SD sequence of the mRNA transcript aligns and binds to the 16S rRNA of the small subunit. this positions the AUG over the P site
pro initiation: what occurs once AUG is over the P site
GTP-bound IF2 binds to the small sub and recruits tRNAfMet to the P site
pro initiation: when tRNAfMet is brought to the P site, is it charged or uncharged
charged
pro initiation: which IF blocks tRNAfMet from entering the A site
IF1
pro initiation: which IF prevents premature binding of the large subunit
IF3
pro initiation: when IF2 brings tRNAfMet to the P site, is it GTP bound or GDP bound
GTP bound
pro initiation: once tRNAfMet is at the P site, what happens
large subunit associates and GTP is cleaved (GTP was bound to IF2), then all IFs dissociate
pro initiation: what is the final product called
intiation complex
pro initiation: describe the initiation complex
AUG over the P site, tRNAfMet over the P site, both subunits intact, no IFs
eu initiation: which IF is equivalent to IF1
eIF1A
eu initiation: which IF is equivalent to IF2
eIF2 and eIF5B
eu initiation: which IF is equivalent to IF3
eIF3
eu initiation: role of eIF2
initial escort of tRNAfMet to the P site
eu initiation: role of eIF5B
GTP hydrolysis
eu initiation: describe the orientation of the 5’ and 3’ ends of the transcript (ie how is this different from pro initiation)
the two ends are linked
eu initiation: what links the 5’ and 3’ end
eIF4F complex
eu initiation: other than the eIF4F complex, which molecule is required for transcript circularization
polyA binding protein must bind to the polyA tail
eu initiation: when does mRNA bind to the small subunit (ie what mediates this process)
mRNA is able to bind to the small subunit due to eIF4F complex
eu initiation: how is the first AUG found
ribosome scans along the transcript for it
eu initiation: what happens once the first AUG is found
large subunit associates
eu initiation: what happens once the large subunit associates
eIF5B enters, hydrolyses its GTP, and most factors leave
elongation: which three EFs are needed
EF-Tu, EF-Ts, EF-G
what are the three substeps of elongation
binding of incoming aminoacyl-tRNA in the A site, peptide bond formation, translocation
elongation: describe the steps of binding of incoming aminoacyl-tRNA in the A site
aminoacyl-tRNA binds to GTP-bound EF-Tu, and all of these enter the A site. GTP is cleaved, Tu leaves (eventually regenerated by EF-Ts)
elongation: describe peptide bond formation
a peptide bond is formed between the first and second amino acids (in A and P sites). fMet in the P site is treansferred to the amino group of the aa in the A site. This = tRNA in the A site with dipeptide and an uncharged tRNA in the P site
elongation: what catalyzes peptide bond formation
23S rRNA within the ribosome
why is the ribosome considered a ribozyme
the 23S rRNA catalyzes peptide bond formation in translation elongation
elongation: chemically, describe peptide bond formation
amine group in the A site amino acid acts as a nucleophile. It attacks the carbonyl of fMet to form the peptide bond
elongation: what can occur after peptide bond formation
ribosome can move one codon towards the 3’ end of the mRNA (translocation)
elongation: describe how translocation occurs
GTP-bound EF-G mimics the structure of EF-Tu complexed with tRNA. It enters the A site and causes a conformational change in the whole ribosome = translocates it downstream
termination: list the stop codons
UAG, UAA, UGA
termination: what happens when a stop codon reaches the A site
release factors help cleave the peptide-tRNA bond, release the peptide, and dissociate from the ribosome
termination: what are the two types of release factors
RF1 and RF2
termination: which stop codons does RF1 recognize
UAG, UAA
termination: which stop codons does RF2 recognize
UGA, UAA
termination: where do RFs transfer the peptide to
a water molecule
termination: what happens after the peptide is transferred to water
RF binds to the A site and mimics a tRNA. the ester linkage between polypeptide and tRNA in the P site is hydrolyzed
termination: what happens once the ester linkage of the polypeptide and tRNA in the P site is hydrolyzed
RF leaves and is replaced by GTP-bound EF-G and ribosome release factor (RRF). GTP hydrolysis releases the large subunit
termination: what happens once the large subunit is released
IF-3 replaces EF-G-GDP, which helps release the tRNA and mRNA and prepares the 30S subunit for the next cycle
describe 2 points in time where protein folding may occur
- some fold as soon as the peptide begins exiting the ribosome
- some require chaperones to fold properly
list 4 additional post-translational modifications a protein might need
removal of fMet, loss of signal sequences, attachment of carbohydrate side chains, attachment of isoprenyl groups
list 3 antibiotics that disrupt translation in bacteria
tetracycline, streptomycin, puromycin
what does tetracycline do
blocks A site
what does streptomycin do
inhibits initiation
what does puromycin do
forms peptide bond and terminates growing polypeptide
list 2 toxins that inhibit translation in mammals
diptheria and ricin
what does diptheria do
inactivates EFs
what does ricin do
inactivates 60S subunit
add: energy cost section
