Lecture 34: RNA Translation Flashcards
genetic code
specifies 64 triplet codons in mRNA that correspond to the 20 amin acids and 3 termination codons
How many nucleotides in the codon:anticodon base pairing are required to specify the correct tRNA
only 2 of 3
what is the thrid nucleotide
in the wobble position
premits noncanonical base pairing (inosine)
how many nucleotides in a tRNA molec
70-90
on an L shape conf
3 basic features of tRNA
1) triplet anticodon (base pairs with mRNA)
2) amino acid attachment site (at 3’ CCA sequence)
3) multiple contact points outside codon recognized by aminoacyl tRNA synthetases
aminoacyl-tRNA synthetases error rate
10-4 of amino acid charging
requires accurate recognition before and after adenylation reaction and proofreading function
charging tRNA
process of linking tRNA and amino acid
MUST ATTACH COVALENTLY
answer 2 questions before we could link RNA to protein synth
1) what molec serves as the molecular “adaptor” between amino acid pool and mRNA?
2) how many nucleotides in mRNA are needed to specify each amino acid?
what was known?
20 aas
GC base pair
A U/T base pair
2) how many nucleotides in mRNA are needed to specify each amino acid?
if codon consisted of 2 nucleotides: 4^2= 16, only 16 amino acids
if 3: 4^3= 64 amino acids. have to go with this because 16 amino acids is too small
How do we know which nucleotide triplets encode which amino acids?
Marshal Nirenberg
start w/ known tRNA sequences
incubate radioactive tRNA-amino acid adaptors w/ cell extract and artifical template
see which combo makes protein
only did: UUU gave Phe, AAA gave lysine, CCC gave Pro (no GGG)
revealed 3 (61 to go!)
what about the other 61 (not UUU, CCC, AAA)
Nirenberg sunthed short RNA templates
incubated w/ known radioactive tRNAs charged w/ amino acid and add ribosomes
identified interactions
determined 54 of the 64 possible codons
the final 10 codons
Gobind Khorana
synthed repeating dinucleotide templates
ID’d corresponding polypeptides
depended on 3 “reading frames”
same sequence, depends on where you start reading
results of the three experiment lead to the…
Genetic Code
exists in all living orgaisms that synth protein from RNA using ribosomes
reading frames
where you start and select units of 3
for the most part…code includes…
61 amino acid specified codons
3 termination codons (found in 3’ region of mRNA coding sequence)
stop codons
NOT recognized by tRNA!!!
Start codon
Met (almost always just a start codon)
Which two amino acids are specified by only ONE triplet codon (see genetic code on slide 7)
Met, Trp
Which three amino acids are specified by SIX triplet codons (see gentic code on slide 7)
Arg
Leu
Ser
What is normally the first codon
AUG (encodes methionine)
How many are recognized by tRNA then?
61 of 64 (stop codons are not!)
So why use the genetic code?
it lets us predict the primary protein sequence from an RNA transcript (or DNA coding strand)
first codon usually AUG
last usually a stop codon
reading frame must be non-overlapping so once ribosome innitiates, protein sequence is fixed
good reading frame
50+ codons w/o stop codon is a good predictor of a protein coding gene
average protein coding sequence for a 30-60 kDA protein requires 350-500 codons!!!!!!!!!!!
are Met only used as start codons?
NO!
Considering that 61 codons specify amino acids and 3 codons are terminators, what is the frequency of a termination codon in a reading frame BY CHANCE?
3/64= 5%
or 1 in 20
Wobble Hypothesis
first 2 nucleotides in codon specify amino acid
THIRD nucleotide in codon is VARRIABLE!
third codon may not have watson-crick base paring… Inosine (I) !!!!!
Why is the third nucleotide variable?
if each codon needed a single tRNA w/ corresponding anticodon, we’d need 61 amino acid codons and 61 different tRNAs!
we only have about 30 tRNA molecs, so some tRNA anticodons MUST recognize multiple codons
Inosine!
tRNA often has this nucleotide substituded in anticodon loop
generated by deamination of adenine (results from tRNA base modification during tRNA processing)
Inosine binding
hydrogen bonds with uracil, cytosine, and adenine
guanine can hydrogen bond with uracil
Back to Wobble hypothesis
3’ nucleotide in codon and 5’ nucleotide in anticodon sequence must pair in uncoventional ways (I)
because of convention of writing in 5’ to 3’ direction
inosine and wobble hypothesis: when inosine is in 5’ position of tRNA anticodon: 3’ amino acid codon in mRNA can be….
U
C
A
and still form H bonds
if 5’ nucleotide in tRNA anticodon is G or U…
it can form H bonds with
U or G (respectively) in 3’ amino acid codon in mRNA
so wobble hypotehsis…
uncoventional base pairing
specifies that 3’ amino acid codon in mRNA is the wobble position
if first base pair of anticodon is… C
third base of codon can be…
G
if first base pair of anticodon is… A
third base of codon can be…
U
if first base pair of anticodon is… U
third base of codon can be…
A
G
if first base pair of anticodon is… G
third base of codon can be…
U
C
if first base pair of anticodon is… I
third base of codon can be…
U
C
A
What are two evolutionary advantages of degeneracy in the Gentic Code (wobble)
fewer tRNAs,
so we dont have to carry around as much genetic material
allows more tolerance for mutations, not every base change would result in a mutation!
STUDY SLIDE 11
STUDY SLIDE 11
so why is the number of tRNA molecules in an organism less than 61?
because dgeneracy allowed by wobble position
however, some amino acids are specified by 4-6 codons
anti codons with a C or A in wobble position…
can only read one codon!
so… what is the minimum number of tRNA molecs required for 61 codons?
32!
tRNA structure
cloverleaf structure (2D)
4 helical regions (extensive RNA-RNA base pairing)
loops
variable arm
Commonalities:
all have CCA 3’ sequences, 3’ OH is where tRNA and amino acids attach covalently (by tRNA syntehtases)
tRNA loop names
DHU loop:
T psy C loop
anticodon loop: needs to be positioned so that bases are flipped out and can interact with outside stuff
(variable extra arm)
loop regions are strucural when we make it 3D
make it into the L structure
tRNA helical regions
stack
modifcation of tRNA molecs
usually 10-15 modified bases
contribute to tRNA substrate binding
tRNA for alanine
4 differnt codons encode it
5’IGC3”
I can recognize 3 of the codons
there is also 5’CGC3’ to recognize the 4th Ala codon
so there are 2 diferent anti codon sequences
Aminoacyl tRNA synthetases
enzymes that attach amino acid to tRNA
20 of them in most organisms (1 for each of the amino acids) may have to recognize more than one tRNA (since there are 32)
very important: if they do it wrong, amino acid gets incorporated into a protein, this is bad
tRNA attachment to amino acid
1) amino acid gets bound to AMP (activated a.a.)
ATP + a.a.= PPi release
2) covalently bind adenylated a.a. to adenosine residue at 3’ end of tRNA, release AMP (released from tRNA?)
3) charged tRNA released
energy cots for aminacyl transfer reaction
2 ATP equivalents
What happens during protein synth in the aminoacyl tRNA synthetase charges the tRNA with thw WRONG amino acid?
we get the wrong amino acid incorporated into a protein
2 important tasks accomplised by covalent attachment of an amino acid to the CCA sequence at the 3’ end of the tRNA
1) links RNA sequence to protein sequence by mathcing tRNA antocodon to right amino acid
2) chemically activiates amino acid to faccilitate peptide bond formation during ribosome-mediate protein synth
more than just the anticodon sequence is checked to make sure the match is right
Amino Acid proofreading in aminoacyl tRNA synthetases… what does aminoacyl tRNA synthease contain
activation site
editing site
at least 2 of three proofreading edits
3 ways to make sure right amino acids are bound (all use 2 of 3)
size
adenylated AA is hydrolyzed if too big
remove wrong amino acid after charging
Edit 1
if amino acid is too big to bind correctly,
Edit 2
PRE TRANSFER EDITING
if adenylated Amino acid is too small to fit in editing spot, it is hydrolyzed
Edit 3
Post transfer editing
remove incorrect amino acid after charging has occured
hydrolyzation by 3’ site, aa and charged tRNA released
which edit MUST take place
edit 1 (based on size) either 2 or 3 can happen
these take nrg, but is important that they happen to avoid errors
practice slide 20
practice slide 20
where is the unconventional base? (wobble)
5’ position of ANTIcodon
where is the unconventional interaction? (wobble)
between the 5’ ANTIcodon and the 3’ codon
other than I, whats the weird base pair
GU base pairs
