Lecture 19

Question 1

One potential example of leMARKian evolution:

Answer 1

one potential example could be the marks (writer/eraser) put on our DNA

possibility for some effects of things that happened in one generation to be passed onto a few of the next generations

Question 2

Translation parts list:

Answer 2

• mrna- the template
• amino acids (20)
• tRNAs (approximately 40)
• ATP
• GTP
• ribosome
• -small subunit (decoding center)
• -large subunit (peptidyl transferase center)
• initiation factors
• elongation factors
• termination factors

Question 3

Codons

Answer 3

First letter of codon is at 5’ end, third letter is at 3’
Start: AUG
Stop: UAA, UAG, UGA
Almost universal but not quite. Mit and chloroplasts have some slight variations. some siliates don’t eve have stop codon (can code for aa or can mean stop)
3 letter code = codon
degenerate = multiple codons for the same thing
leucine has 6
20 aa and only four bases, from the beginning knew it had to be at least a 3 letter code, maybe more
notice: all non polar aa on the left side of the table
almost all polar and charged guys on the right side- not an accident
64 possibilities, w/ only 20 aa
a bunch coding for the same thing
if you got deamination of cytosine, (common) (transitions)
would just move up and down the table, not across
most common mutations change a polar thing for a polar thing

Question 4

Genetic code

Answer 4

The set of rules by which a linear sequence of nucleotides specifies the linear sequence of a polypeptide

Question 5

What is the nature of the genetic code?

Answer 5

Figured out the code must not be overlapping
was continuous
if you had a mutation with an overlapping code, one point mutation would affect three amino acids

Question 6

Reading along a DNA or RNA strand in one direction there are three possible reading frames

Answer 6

can shift this reading frame
note: in ds DNA there are six possible reading frames: three on bottom and three on top strand
a +2 change in reading frame is equivalent to moving the frame -1

Question 7

Understanding the genetic code

Answer 7

If you get a +1 nt insertion, and combine with a -1 close by, you can fix it
you get a little garbage, but everything else restored (restorative power) garbage in the middle- part they messed up was not important to function
+1 and minus and +2 and minus helped them understand the reading frame
Figured out it was a multiple of three
were able to figure out basic properties of genetic code
certain +1s and certain -1s could not be fixed
mess up a critical part of the protein, even when you restore it protein won’t work
how they learned about stop codons

Question 8

Figuring out the coding table

Answer 8

took about 20 more years
2 types of experiments used
1) synth polymers of rna nucleotides. could not control what seq was but could take rna and make proteins. just uracil = UUU = phe. put a couple in a tube and used probabilities
2)synth 3 bp nucleotides (ACA) put their tRNAs for all aa and find out what would pair

Question 9

Codon assignments

Answer 9

Genetic code evolved and was selected for
based on this, we like leucine (v hydrophobic and valuable
whereas methionine and tryptophan are used more rarely

Question 10

Codons again

Answer 10

notice: not all non polar aa on left side of the table
almost all polar and charged guys on the right side
not an accident
if you got deamination of cytosine (rel common) (transition) would just move up and down the table, not cross
most common mutations change one polar thing for another polar thing

Question 11

Who came up with the idea that you would need an adapter?

Answer 11

francis
never published
the adapter is tRNA

Question 12

Crick: adaptor hypothesis

Answer 12

gonna need an adaptor to do base pairing
provides specificity
have amino acid up top, with amino acid binding site, adaptor, and adaptor binds to nucleotide triplet coding for an amino acid

Question 13

tRNA: the adaptor

Answer 13

t is for transfer
small rna, 80 bases long
have a base paired point and a loose, non base paired part
three bases will pair
have an anti codon on tRNA that matches with codon on mRNA
3’ end is where you have an amino acid

Question 14

tRNA

Answer 14

sticking aa on up here, but codon is down there
forms an L shape
how does an enzyme know what to put there based on what’s going on way down there, plus all aa look really similar
answer: also information along stems and loops
there’s a t for thymine, psi is a modified uracil (in the tRNA)
tRNAs have all these bases that have been modified after synthesis
lots of loops and bulges
D arm contains two or three D residues at different positions
Wobble position is the first one: the 5’ end of the anticodon

Question 15

RNA secondary structrue

Answer 15

has double helices similar to A DNA

Question 16

Wobble

Answer 16

Often a whole bunch of guys in a single box will be the same but differ in the third letter
crick figured maybe different anticodons don’t have to hybridize perfectly
third position can tolerate some imperfection

Question 17

Wobble: alanine tRNA recognizes GC (A/C/U) codons

Answer 17

5’ pos of anticodon (3rd position for mRNA) can have a lot of play in pairing
ex: use I for inosine (step where you could either synth guanine or adenine)
can pair with either C or U, or with A as well

but still have problem of the chemistry: knowing what’s down here pairing with mRNA, and sticking the aa on here
step where translation takes place

Question 18

Wobble examples

Answer 18

1) one codon recognized
A/U and C/G
2) two codons recognized
U recognizes A or G, G recognizes C or U
3) three codons recognized
I recognizes A, U, or C
problem: for how many tRNAs do we need one codon? just one because of inosine
can pair with all three, so one tRNA will get the job done
needs to be at least 31 different tRNAs
humans have 150

Question 19

How inosine promotes wobble pairing

Answer 19

base of inosine is hypoxanthine
inosine is adenine deaminated
can bp with cytosine, uracil, and adenine

Question 20

Differences between tRNAs

Answer 20

3’ OH acceptor site can be in different places
enzymes figure out info all along from different bulges, plus anti codon, what we should put up here
look similar but there are slight differences

Question 21

tRNA synthetases assign the codons

Answer 21

aaRS = aminoacyl tRNA synthetase
enzymes that figure out the chemistry
only one of these for each aa, with one exception
3 diff tRNAs all recognized by the same one of these
way these enzymes bind is different in each place
critical for getting translation done correctly

Question 22

distribution of bases required for correct charging of tRNAs

Answer 22

the diameter of each yellow circle indicates the number of different tRNA molecules for that position is an aaRS specificity determinant
can’t only use info just at stem, need it all along

Question 23

2 classes of amino acyl tRNA synthetases

Answer 23

classes are in terms of chemistry- get the gist

Question 24

activation of amino acid and formation of charged tRNA

Answer 24

tRNA synthetase adenylates an amino acid using ATP, releases pyrophosphate
then tRNA synthetase takes adenylated amino acid and tRNA and makes aminoacyl tRNA, releasing AMP
activate for more chemistry
get pyrophosphate to drive reaction forward (common)

where did chemistry look like this? fatty acid oxidation. adenylate a fatty acid and transfer acyl adenylate to coa and starts oxidation
same enzyme does both steps
charged tRNAs have an amino acid

Question 25

activation of amino acid

Answer 25

nucleophilic attack by carboxyl group of aa
get int
can do one of two things here: either attach to 2’ or 3’ hydroxyl group
get 5’ aminoacyl adenylate (aminoacyl-AMP) with mixed anhydride

Question 26

Formation of charged tRNA (I)

Answer 26

does not matter which hydroxyl, either way will get transesterification w/ 3’ hydroxyl on the end
Class I aminoacyl-tRNA synthetases put on 2’
Class II on 3’

Question 27

activation of amino acid energy use

Answer 27

uses two Pi’s plus heat
use a lot of energy in this process to get it right
forming peptide bond actually takes a lot of energy too- not favorable

Question 28

Three sources of error in translation

Answer 28

all tRNAs look really similar
pick wrong one: get wrong aa on tRNA
also, aa look similar, pick right tRNA but might get wrong aa
even if right aa on right tRNA, ribosome might pair with wrong codon and you’d get wrong aa
1) aarRS uses wrong aa as substrate
2) aaRS uses wrong tRNA as substrate
3) ribosome selects wrong aa-tRNA for codon

Question 29

Example: why it’s hard to achieve specificity in aaRS

Answer 29

two amino acids that are really similar 
valine and isoleucine
not a big diff in free energy
at eq, happens 1/200 times
lots of mistakes
actual error rate for protein synth is 100 fold lower
enzymes will do proof reading
similar to proof reading with pol