Download Protein Synthesis I

FUNDAMENTALS I: 10:00-11:00
9-15-2010
MILLER
I.
PROTEIN SYNTHESIS I
Scribe: LOUISA WARREN
Proof: CHRISTINE SIRNA
Page 1 of 5
PROTEIN SYNTHESIS [S1]:
a. This process is called translation, which is sometimes compared and contrasted to transcription
i. Transcription is when DNA is used to make an RNA molecule
ii. In transcription, there is no new element involved except for the change in oxygen (deoxy vs oxygenated
molecules). It is generally a copying process - nothing new is done.
iii. In translation, something that is 2D in one chemical is eventually transformed into something that is 2D in
another chemical (amino acids) that are able to form all types of different structures (primary, secondary,
tertiary, quaternary structures of proteins)
b. Nucleic acid polymers generate amino acid polymers (proteins) that can form all of these structures
c. Translation into another language is a tough process – the chemical process is even more difficult
d. 4 major players in protein synthesis:
i. Messenger RNA’s- the result of transcription of a DNA segment
ii. Transfer RNA’s
iii. Aminoacyl tRNA synthetases
1. Remarkable enzymes that make translation possible
2. Unique- there are not many- there is one synthetase for each amino acid
3. Called synthetases because they have ATP involved in their activity
iv. Ribosomes
1. Large structures
2. Combinations of proteins and nucleic acids in definitive array
3. Where coupling between amino acids and protein takes place
II. GENETIC CODES AS THEY OCCUR IN mRNA’S [S2]
a. mRNA is a message that contains the codes for the amino acids
b. Codes can be very different- you can have a nucleic acid polymer which has a code at every segment of the
nucleic acid
i. For instance, you could start synthesizing a protein with the three nucleotides in the beginning, or you could
start with three nucleotides starting with the second nucleic acid
1. That is the process used by small bacteria, viruses, and other organisms
2. They like to synthesize quite a few proteins but have only a small code/ small mRNA
3. We do not have that- we have plenty of room for a large amount nucleic acid, so we use 3
nucleotides for each code with no overlap
c. We use the first 3 nucleotides, then the next three, the next three, etc.
i. It takes 3 nucleotides to make a code for one amino acid
d. The code is continuous in mammal cells, but the message has to be prepared for that
i. Initially synthesized messages are spliced to make mature messages
ii. That takes out the commas and spaces to leave us with a continuous message
iii. If there is an interruption in the message, it is generally fatal for the message
e. So we have a continuous, non-overlapping code where every 3 nucleotides code for one amino acid
f. Each sphere in the figure is one nucleotide
III. GENETIC CODES [S3]
a. Since we have 4 nucleotides and 3 in every code, there is the possibility for 43= 64 different codes
b. We only have 20 amino acids, so there is a great deal of redundancy- there many codes for one amino acid
for most amino acids
c. All 64 codes listed here can be divided into various segments
i. Grey areas- 3rd nucleotide can be either U, C, A, or G
1. So the 3rd nucleotide for leucine doesn’t matter- it can end in U, C, A, or G
2. 8 families where the 3rd position has very little relevance
ii. Beige- 3rd position can be either A or G
1. 6 families
2. For example, leucine can be coded by UUA or UUG
3. There are other amino acids like that: glutamic acid, lysine, glutamine, and arginine
iii. Yellow- 3rd position can be either U or C
1. 7 pairs
iv. Dark blue- 3 out of 4 nucleotides are used for isoleucine
1. They can be U, C, or A (not G)
2. Only situation like this
v. Dark yellow- the unique ones
1. AUG for methionine
FUNDAMENTALS I: 10:00-11:00
Scribe: LOUISA WARREN
9-15-2010
Proof: CHRISTINE SIRNA
MILLER
PROTEIN SYNTHESIS I
Page 2 of 5
2. UGG for tryptophan
3. Methionine and tryptophan are the only amino acids where there is only one code
4. Methionine and tryptophan are the least used amino acids in all proteins
vi. Stop codons
1. Example: UGA
2. When that codon is read, enzyme stops translation and essentially cuts off the protein so no new
amino acid is added to the growing polypeptide chain
d. The looseness of these codes is remarkable, but there is a reason we will discuss
e. This is classic redundancy
i. You would call this degenerate because it is not precise
ii. That is why the genetic code is called degenerate- it is not precise but can be modified based on the needs
of the organism
f. These codes reside in the mRNA
g. All 64 codes are utilized to code for some amino acid except for the 3 stop codons
i. So out of the 64 total codes, 61 are utilized to place an amino acid
IV. THE GENERAL STRUCTURE OF tRNA MOLECULES [S4]
a. tRNA’s are small segments of nucleic acids- they never go beyond 75-85 nucleotides
b. Generally pictured in this clover-leaf arrangement
c. Two traits of every tRNA:
i. The 3’ end is the business part- where the amino acid will be attached to tRNA
ii. The base at the end of the 3’ will always be Adenine
d. All of the other locations can be changed
e. There are loops:
i. Thymidine, pseudouridine, cytosine loop contains those three nucleotides
ii. Variable loop- dots show that there can be 1-8 nucleotides in loop, and it is highly variable
iii. Anticodon loop- where tRNA recognizes the message
iv. D-loop- has dihydrouridine in it
f. All of these locations are very specific, but every tRNA has a signature
i. tRNA is usually designed to carry just one amino acid, but the signatures for tRNA’s designed to carry the
same amino acid can be very different- degeneracy is all around us
1. For example, a tRNA designed to carry alanine can have a different signature from another tRNA
designed to carry alanine
ii. There can be a lot of tRNA’s for one amino acid, and the synthetase recognizes all of them in the correct
way
1. For example, there are 45 tRNAs carrying alanine, and the synthetase designed to place alanine on
tRNA will recognize all of them- that is why synthetases are such marvelous enzymes
g. Remember that there are various points along this molecule where its signature can be expressed
V. RIBBON DIAGRAM OF tRNA TERTIARY STRUCTURE [S5]
a. tRNA does not really have that clover leaf appearance but looks more like a post with a handle on it
i. It is like where the mailbag was hung on a train so the train didn’t have to stop
b. Adenine is at the 3’ end with the amino acid attached
c. Amino acid is held out to be added on to growing polypeptide chain
d. Nucleic acids are read from 5’ to 3’, so 5’ is positioned at the 1 (read numbers in picture)
e. Notice that the T, pseudouridine, C loop that was on the right has been wrenched to your left, along with the
variable loop
f. The D loop has been brought from left over to the right side
g. The 3’ end and the anticodon are also skewed over to the right
h. Various designations shown on the figure
i. Single letter notations for amino acids (listed at position 73 box) - this is telling you that there are signature
markers for these amino acids located at this position on the tRNA molecule
1. Markers that indicate what amino acid should be placed on there are located all over the tRNA
molecule
2. For example, the variable loop has markers for serine and tyrosine
VI. HOW IS AN AMINO ACID MATCHED WITH ITS PROPER tRNA? [S6]
a. There is only one synthetase but many tRNAs for each amino acid, so synthetase has a remarkable job
b. It is called the “2nd genetic code”- tRNA interprets the genetic code based on what the synthetase does
c. Synthetase for any amino acid has to see the right tRNA and the right amino acid
i. Choice of the right amino acid is easier than choice of tRNA because there may be hundreds of tRNA
around and only 20 amino acids
FUNDAMENTALS I: 10:00-11:00
Scribe: LOUISA WARREN
9-15-2010
Proof: CHRISTINE SIRNA
MILLER
PROTEIN SYNTHESIS I
Page 3 of 5
ii. Synthetase makes the right tRNA charged with the right amino acid
VII. tRNA- SYNTHETASE [S7]
a. tRNA (purple) hugs the surface of the synthetase with the arm going deep into synthetase
b. The amino acid (green) is attached to the tRNA
c. This is a very intimate relationship
d. Three different entities have to be brought together in the proper way with the correct tRNA and amino acid
selected
e. Synthetase has ability to “edit” the results
i. Once the amino acid is hooked on to the tRNA, is it the right one?
ii. Example of when synthetase could be confused between 2 amino acids: valine and isoleucine (differ by
one methyl group on the side)
iii. Only about ½ of the proteins we make get folded- one reason is that there is a 10-20% failure of
synthetases to put the right amino acid on the right tRNA
1. So not all proteins are synthesized properly
2. That is one reason why it is more economical to make quaternary structures out of small numbers of
amino acids rather than one huge protein with a lot of amino acids
a. You can make ten 100-amino acid peptides more easily than one 1000-amino acid peptide
because of the mistakes that can be made
b. Not even protein synthesis is perfect
VIII. MAJOR IDENTITY ELEMENTS IN 4 tRNA SPECIES [S8]
a. The markers on a tRNA are variable and can be found all around the tRNA
b. Yeast tRNA charged with phenylalanine
i. Marker near the stem where the amino acid will be attached
ii. Markers in the D loop and in the anticodon
c. Methionine tRNA
i. Marker is in the anticodon region
ii. Methionine is one of the 2 amino acids that have only one code, so you would expect the marker for that
tRNA to be in the anticodon
d. Serine tRNA
i. Markers all in the stem at the 5’ and 3’ sides and in the D loop
ii. Notice large variable loop
e. Alanine tRNA
i. Markers in the stem region but not as prevalent as in serine tRNA
ii. Small variable loop
f. Those signature locations are read by synthetase and allow synthetase to accept the tRNA
IX. THE AMINOACYL-tRNA SYNTHETASE REACTION [S9]
a. Charges tRNA with the amino acid
b. Top of the figure: overall view of the reaction
i. You have the amino acid and the tRNA (left)
ii. ATP is ultimately converted to AMP and pyrophosphate
1. The carboxyl will make an attack at the 2nd phosphate of AMP
2. The first phosphate is esterified to the ribose, but the second and third phosphates are connected by
anhydride bonds
3. Anhydride bonds are high-energy bonds that release energy when hydrolyzed
a. The hydrolysis reaction goes very quickly because it is going from a higher to a lower energy
state
iii. The amino acid is ultimately esterified to a tRNA (product on right)
iv. The overall reaction is driven by the utilization of ATP, cleaved at the 2nd phosphate region, and then the
cleavage of pyrophosphate
1. Cleavage of 2 high-energy bonds is used to charge the tRNA- an expensive process
c. Specifics
i. Enzyme (blue dot), ATP, and amino acid are used to make an AMP derivative of the amino acid, and
pyrophosphate is given away
1. AMP is linked to the amino acid with a mixed anhydride bond- bond between a carboxyl carbon and
a phosphate acid
2. This is a powerful bond, but it has taken 2 high-energy bonds to make
a. Broke 2 anhydride bonds from ATP to form 1 mixed anhydride bond
i. This is the usual result- there is no conservation of energy
FUNDAMENTALS I: 10:00-11:00
Scribe: LOUISA WARREN
9-15-2010
Proof: CHRISTINE SIRNA
MILLER
PROTEIN SYNTHESIS I
Page 4 of 5
ii. A hydroxyl group on the ribose of the last nucleotide on tRNA makes a nucleophilic attack on the mixed
anhydride to capture the carbonyl carbon and derivatize the amino acid with an ester bond to the ribose
moiety of the tRNA
1. tRNA has an adenine at the 3’ position, so the ribose of the adenine captures the amino acid of the
ester bond
2. This reaction works because going from a high-energy mixed anhydride bond to an ester bond
a. It will later go from an ester to a peptide bond
3. Whole thing works because it goes from high-energy ATP, to mixed anhydride, to an ester bond that
attaches the amino acid to tRNA
4. This reaction is done with class I synthetase
a. Imperfect reaction because the amino acid needs to be on the 3’ hydroxyl of the ribose, so
there is a transfer to move the ester bond from the 2’ carbon of ribose to the 3’ carbon
i. Transfer is a slight expenditure of energy
b. This results in the same type of derivative, just transferred from one carbon to another
iii. Class II is easier
1. Same type of reaction occurs, but the attack is done from the 3’ ribose hydroxyl to the carboxyl
carbon of the amino acid
2. The amino acid is esterified directly to 3’ carbon of ribose moiety rather than having to switch from
the 2’ to the 3’
d. Once these reactions have occurred, tRNA is “charged” with the amino acid
e. This charging process is the same reaction for every tRNA and every amino acid- the only difference is which
amino acid go to which tRNA, which is highly degenerate
X. CODON-ANTICODON PAIRING [S10]
a. How the tRNA and the message get together
b. Message from transcription of DNA is shown from 5’ to 3’ and labeled as amino-terminus to C-terminus to
show that the first amino acid is synthesized at the amino terminus, which is started at the 5’ end
i. Amino acids are added to the carboxy terminus by the time you get to the 3’ end of the message
ii. Peptide is synthesized from the amino terminus to the C terminus and the message is read from 5’ to 3’
c. When nucleic acids come together, they are always antiparallel
i. 5’ to 3’ position of the mRNA lines up with the 5’ to 3’ of the tRNA in the other direction
ii. tRNA comes in antiparallel to the message so the 1st position of the anticodon connects with 3rd position of
codon
iii. 2nd with 2nd and 3rd position of anticodon with 1st of codon
iv. Comes in “backwards”
d. Redundancy was in the 3rd position of the codes and 1st position of anticode
i. This is called the wobble position
ii. The codes vary dramatically in their third position
iii. This position is very unstable, and the interaction between these two bases is very flimsy
iv. The number of tRNAs for each amino acid is not the same as the number of codons because some
anticodons can recognize numerous codes
e. Specificity is determined by the first two bases of the code and 2nd and 3rd bases of the anticodon
i. Flexibility and speed come from the wobble position
XI. WOBBLE POSITION RULES [S11]
a. First two bases of the codon form strong Watson-Crick base pairs with corresponding bases of the anticodon.
The third base in a codon pairs loosely with the corresponding base of its anticodons.
i. 3rd base in codon has a flimsy interaction with the1st base of the anticodon
ii. 2-2 and 1-3 bonds are very good
b. If first base of anticodon is C or A, binding is specific because of strength of G-C pairs and A-U pairs
i. Strict specificity when first base is C or A
c. If first base of anticodon is U or G, binding is less specific and two different codons can be read by that tRNA.
i. Loose connections when first base U or G
d. If first base of anticodon is Inosine, binding is less specific and three different codons can be read
XII. ILLUSTRATION OF RULES [S12]
a. Top row: If C or A in the first position of the anticodon, strict binding
b. Middle row: If U or G in the first position of the anticodon, binding can be either with A or G (with U) or C or U
(with G), weak binding
c. Bottom row: If Inosine is in the first position of the anticodon, binding will be very weak- A, U or C can all be
recognized
FUNDAMENTALS I: 10:00-11:00
Scribe: LOUISA WARREN
9-15-2010
Proof: CHRISTINE SIRNA
MILLER
PROTEIN SYNTHESIS I
Page 5 of 5
d. When you want to speed up protein synthesis, you need tRNA to be expelled quickly from transfer machine
once it has been used
i. When tightly bound, it takes longer to expel than if it is bound in this way
ii. When rapid protein synthesis is needed, use an anticodon that has a U, G, or I in the first position
1. Organisms do this without thinking about it
2. One of the rationales for this situation
[End 46:45 mins]