Protein Synthesis

Question 1

What are the key features of the Genetic Code?

Answer 1

1) The genetic code is universal: All organisms use the same genetic code
2) The genetic code is degenerate: Multiple codons encode for the same amino acid. e.g. GGU, GGC, GGA and GGG all encode for glycine. Degeneracy is usually at the third nucleotide or the wobble base.
3) Some codons have multiple functions: AUG for example encodes for Methionine but it is also the START codon. UAA, UAG, and UGA all are STOP codons.

There are 64 total triplets of nucleotides but only 20 amino acids that are coded for so we clearly have more than one triplet coding for a specific amino acid.

Codons for the same amino acid tend to share the same first two nucleotides (but this is not always true). Also, not every amino acid has more than one codon that codes for it.

Question 2

Identify features of an mRNA that can affect its translation.

Answer 2

An mRNA needs to contain a 5’-cap and a Polyadenylated tail in order to be read as a “good” mRNA. Also, the mRNA must have the correct reading frame. The sequence of nucleotides are read 5’ to 3’ in translation in consecutive sets of 3 nucleotides. Thus, there are hypothetically 3 reading frames that exist and thus the same mRNA could give 3 different polypeptides. In reality, only one of these reading frames contains the actual message and if the wrong reading frame is read, there tends to be a lot of sporadic STOP codons.

Question 3

Translation START codons

Answer 3

AUG

Codes for Methionine

Question 4

Translation STOP codons

Answer 4

UAA, UAG and UGA

They do NOT code for amino acids.

Question 5

Degenerate

Answer 5

Multiple codons code for the same amino acid

Question 6

4 Major Steps in Translation

Answer 6

1) Charging of the tRNA: Placing of the amino acid
2) Initiation
3) Elongation
4) Termination

Question 7

Transfer RNA (tRNA)

Answer 7

It is synthesized by RNA Pol III. Also, it is synthesized as a precursor, it will be trimmed, spliced, and modified. There are over 50 different modifications for tRNA and one out of every 10 bases will be modified. This modification is thought to be important for the final folding of the tRNA and to get the anticodon region in the right position to get good base pairing with the codon.

tRNA contains some unusual bases that are created during the modification of it such as pseudouridine and dihydrouridine which are derive amino acids from uracil.

There are 2 areas of interest:

1) anticodon
2) 3’ acceptor stem

Question 8

Anticodon

Answer 8

it is a region on the tRNA that is 3 nucleotides long and is responsible for base pairing with the codon to ensure correct amino acid addition to the newly synthesized polypeptide chain. The pairing proceeds from the 5’ end of the codon. Once the first two positions are paired, the exact base pairing of the third (wobble position) is less critical. Wobble positions allow some tRNAs to recognize more than one mRNA codon making it possible for less than 64 tRNAs to recognize all 64 codons. The wobble position in terms of the tRNA though is in the FIRST position, but we call it the third position in regards to the mRNA.

READ mRNA 5’ to 3’ NOW!!!

Question 9

3’ Acceptor Stem

Answer 9

This is where the amino acid is physically attached to. Aminoacyl-tRNA synthetase catalyzes the two step activation reaction of the tRNA.

Question 10

Aminoacyl-tRNA Synthetase

Answer 10

Catalyzes the two step activation reaction of the tRNA. It catalyzes the addition of the amino acid to the 3’ acceptor site. Eukaryotes have a DIFFERENT aminoacyl-synthetase for EACH amino acid whereas in prokaryotes, each synthetase couples more than one amino acid. The aminoacyl-synthetase is also crucial in regards to proofreading. It is the last check to make sure that the correct amino acid was added to the tRNA.

Question 11

Explain the differences between prokaryotic and eukaryotic cells with respect to aminoacyl-synthetases.

Answer 11

Eukaryotes have DIFFERENT aminoacyl-synthetases for EACH amino acid.

Prokaryotes have ONE synthetase which couples for more than one amino acid.

Question 12

Identify important structural features in tRNA molecules, including features that contribute to fidelity in protein synthesis.

Answer 12

Two major structural features are:

1) anticodon
2) 3’ acceptor site

the anticodon ensures correct base pairing with the codon sequence of the mRNA, specifically with the first two nucleotides. It is what reads the codon to see what amino acid is supposed to be added. If the sequences don’t match, they will not bind which gives it fidelity.

The 3’ Acceptor site adds to the fidelity because it binds specifically to the correct amino acid via the assistance of the Aminoacyl-tRNA Synthetase. The specific aminoacyl-tRNA Synthetase has an editing site that ensures the correct amino acid is bound.

Question 13

Draw an aminoacylated tRNA molecule and diagram the reactions that produced it.

Answer 13

View reaction in handout. IT IS A 2 STEP ACTIVATION OF THE tRNA. Basically, carboxy-end’s OH reacts with ATP, adding AMP to the O, losing 2 inorganic phosphates and the H. Then, the now adenylated amino acid (AMP-Amino acid) reacts with the OH of the 3’ acceptor site, giving off AMP and resulting in the amino acid bound to the tRNA via a high energy ester-linkage, thus activating it. This energy is important because it plays a crucial role in providing the energy to create the polypeptide chain. Overall it is using the energy from ATP hydrolysis to add the amino acid to the tRNA.

Question 14

Explain the role of editing by some aminoacyl-tRNA Synthetases in determining the fidelity of protein synthesis.

Answer 14

The synthetase is actually sensing that the correct amino acid is bound to the correct tRNA. It plays a crucial editing step to ensure high fidelity because it is one of the only steps to do this. The synthetase is able to do this so well because it is physically interacting with both the amino acid and the tRNA. It is interacting with the various parts of the tRNA and will make sure the 3 anticodons are correct and match this amino acid. However, some anticodons that code for the same amino acid are very different so it also relies on other parts of the tRNA as well.

Question 15

What are the two “adaptors” that the genetic code is translated by means of?

Answer 15

1) The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA
2) The second adaptor is the tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA.

These two occur directly after one another and an error i neither step would cause the wrong amino acid to be incorporated into the protein chain.

Question 16

“Second Genetic Code”

Answer 16

The recognition of the correct tRNA by the aminoacyl-tRNA Synthetase

Question 17

Aminoacyl-tRNA Synthetase and tRNA interactions to ensure correct tRNA and amino acid binding

Answer 17

The aminoacyl-tRNA Synthetase recognizes multiple regions on the tRNA to ensure that it is indeed the correct amino acid that is binding. The 2 most important and most commonly used regions are the anticodon loop and the 3’ acceptor stem. These are particularly important in controlling the specificity of the aminoacylation reaction. However, some aminoacyl-tRNA synthetases use other regions on the tRNA to distinguish some tRNAs that are difficult to tell apart. There are many regions on the tRNA that the synthetase recognizes that are crucial for this high fidelity.

Question 18

Explain the Proofreading activity of the aminoacyl-tRNA Synthetase

Answer 18

The aminoacyl-tRNA synthetases that need to distinguish between similar amino acids have an added proofreading mechanism to ensure they attach the correct amino acid to the correct tRNA. The correct amino acid has the highest affinity for the synthesis site, however, sometimes errors occur. Thus, the aminoacyl-tRNA synthetase has an editing site in which if the WRONG amino acid has been bound to the tRNA, it will enter the editing site. Then, it will be cleaved off and the correct amino acid will then be added. If the amino acid is correct, it cannot enter the editing site. The accuracy is 1 in 40,000 and this is where most of the proofreading is because once the amino acid is added, there is no going back.

Question 19

Shine-Delgarno Sequence

Answer 19

This is found in PROKARYOTES. It is in front or upstream of the AUG start codon. NOT ALL AUGs HAVE THE SEQUENCE IN FRONT OF THEM. it is a 3-9 PURINE RICH base pair sequence that is just upstream (~10 nucleotides) of the START codon (AUG) and the small ribosomal subunit base pairs directly with it and then the ribosome assembly takes place at the AUG start codon. The shine-delgarno sequence is complementary to the 3’ end of the 16S-ribosomal RNA in the prokaryotic 30S ribosomal subunit. The AUG begins in the P-site of the ribosome and this is where the first tRNA will begin at to give the correct reading frame. fMet-tRNA or formyl-Methionine is what binds first to the AUG and it is a special initiation tRNA with a modified methionine. It is only found in prokaryotes.

Question 20

List the major steps in protein synthesis

Answer 20

1) Charging of the tRNA
2) Initiation
3) Elongation
4) Termination

Question 21

Describe the interaction between the mRNA codon and the tRNA anticodon paying particular attention to the 5’ and 3’ ends of both strands

Answer 21

The mRNA is being read 5’ to 3’, thus, the tRNA’s anticodon will bind to the mRNA antiparallel (3’ with 5’ and 5’ with 3’). first site of the tRNA is the 3’ end of the tRNA, which binds to the first nucleotide (5’ end) of the codon. Then the second site binds to the middle of the codon and the last site, the 5’ end of the tRNA and the 3’ end of the mRNA is the wobble position.

Question 22

Describe the Wobble Phenomenon and explain its relationship to the existence of degeneracy in the genetic code.

Answer 22

The wobble phenomenon basically states that the third nucleotide or the third position of the codon (first of the tRNA when reading it 5’ to 3’) can vary, or “wobble”. In other words, the nucleotide itself can vary from what it should be to still give the correct amino acid since most of the base pairing is mainly dependent on the first two codon positions. In other words, in positions 1 and 2, conventional base pairing MUST take place (A with U and C with G) but in the third position we don’t need this. This relates to degeneracy because when one looks at most codon sequences for amino acids, the first two positions of the codon are mainly the same and the third position is the difference.

Question 23

Describe the functions of the large and small ribosomal subunits in protein synthesis.

Answer 23

The small ribosomal subunit is responsible for establishing the reading frame and helps to initiate protein synthesis. Then, the large ribosomal subunit will come it and it is what has the catalytic activity to start forming the polypeptide chain and forms the peptide bond.

Question 24

What are the initiation steps in prokaryotes?

Answer 24

1) IF-3, the 30S ribosomal subunit (small subunit), and the mRNA come together with the small ribosomal subunit binding to the shine-delgarno purine rich 3-9 nucleotide sequence, placing the ribosome ON the AUG start site (will be in the P-site of large subunit)
2) IF-2 with a bound GTP and the initiator tRNA with a formyl-methionine will come together at the AUG start site. The energy from the bound GTP is critical for initiation.
3) tRNA then binds to the AUG with the IF-2-GTP. The GTP is then hydrolyzed and this recruits the 50S large ribosomal subunit.
4) Elongation occurs

KNOW IF-2 HAS GTP BOUND TO IT

Question 25

What are the initiation steps in eukaryotes?

Answer 25

1) The small ribosomal subunit binds to the initiator tRNA in the P-site (which has a normal methionine) away from the mRNA. The initiator tRNA also has a bound eIF2-GTP (eIF2 has a bound GTP which is critical)
2) The mRNA had eIF4F (eIF4A, eIF4E and eIF4G) which binds to the 5’ methylguanosine cap. This is checking to make sure the mRNA is intact mRNA. If it is not intact it is sent for degradation.
3) The small ribosomal subunit then is recruited due to the eIF4F cap binding complexes being present. The small ribosomal subunit comes and interacts with the eIF4E and eIF4G that are bound to the 5’ cap. It will then bind to the mRNA and begin scanning for the first AUG. IT IS NOT ASSEMBLING ON THE INITIATION CODON!! The eIF4A and eIF4B facilitate unwinding of the mRNA secondary structure.
4) Once the AUG start site is found, the GTP on the eIF2 is hydrolyzed, allowing eIF2 to dissociate from the tRNA.
5) Now the 60S large ribosomal subunit binds to form a complete ribosome ready for protein synthesis. The initiator tRNA will begin in the P-site.

The rate limiting step of the reaction is the reactivation of the eIF2 +GDP –> eIF2 + GT. This is because the eIF2 + GDP needs to be recycled after it is used and it does this using a guanine nucleotide exchange factor, replacing GDP with GTP.

Question 26

Describe the reactivation of the eIF2

Answer 26

The rate limiting step of the reaction is the reactivation of the eIF2 +GDP –> eIF2 + GT. This is because the eIF2 + GDP needs to be recycled after it is used and it does this using a guanine nucleotide exchange factor, replacing GDP with GTP.

If the cell is stressed, eIF2 will become phosphorylated on the alpha subunit and bind irreversibly to the eIF2B that helps catalyze its reactivation. This can lead to protein synthesis ending.

Question 27

Explain how translation is terminated

Answer 27

The ribosome will continue to scan, reading the codons, however, when it reaches a STOP codon in the A-site, a RELEASE FACTOR will come in instead of another tRNA and it will bind to the A-site. Then, the release factor will recruit water when it binds to the A-site and when this occurs, hydrolysis takes place, cleaving the polypeptide from the tRNA in the P-site. Then, the ribosome will disassemble from the mRNA. The release factor looks very much like a tRNA and this is an example of molecular mimicry. This is why the release factor can come and fit into the A-site of the ribosome, interacting with it and the polypeptide to catalyze the addition of water.

Question 28

What are the steps of Elongation?

Answer 28

Each amino acid added to the growing end of a polypeptide chain is selected by complementary base pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain. Because only one of the many tRNAs can base pair with each codon, the codon determines the specific amino acid to be added to the growing polypeptide chain. It is a 4 Step cycle that is repeated over and over again.

1) An aminoacyl-tRNA molecule binds to the vacant A-site on the ribosome by base pairing with the codon of the mRNA.
2) A new peptide bond is formed by the large ribosomal subunit, attaching the polypeptide chain to the amino acid in the A-site.
3) The large ribosomal subunit translocated relative to the small subunit, leaving the two tRNAs in hybrid sites: The P-site on the large subunit now and the A-site on the small subunit still for one and the E-site on the large and P-site on the small for the other. The peptidyl-transferase catalytic activity of the large ribosomal subunit is accompanied by conformational changes that shift the 2 tRNAs into the E- and P-sites.
4) The small ribosomal translocates carrying its mRNA a distance of three nucleotides through the ribosome. This “resets” the ribosome with a fully empty A site, ready for the next aminoacyl-tRNA molecule to bind. In other words, additional conformational changes move the mRNA three nucleotides so the ribosome is reset and ready for the next tRNA. The large subunit moves, and then the small.

Keep in mind that the mRNA is being read 5’ to 3’, synthesizing the new peptide chain N-terminus to C-terminus, thus the amino end of the amino acid is added to the carboxy end of the growing chain.

Question 29

What are the 4 ribosome binding sites?

Answer 29

1) A-site
2) P-site
3) E-site
4) mRNA site

Question 30

Explain the role of GTP hydrolysis in elongation.

Answer 30

There are two elongation factors that play a role in elongation and have vital roles proofreading. EF-Tu and EF-G.

1) EF-Tu: EF-Tu provides opportunities for proofreading of the codon-anticodon match. In this way, incorrectly paired tRNAs are selectively rejected and thus the accuracy of translation is improved. EF-Tu has a bound GTP. This is the last “check” of the amino acid-tRNA combination. EF-Tu has a greater affinity for the tRNA with the correct amino acid bound to it than the one with the incorrect amino acid. Thus, it binds to that tRNA and carries it to the A-site. Then, the anticodon attempts to bind to the codon. The correct match here also has the greatest affinity. Then the GTP on EF-Tu is hydrolyzed causing it to dissociate. EF-Tu introduces 2 lags into the system: 1) When GTP is hydrolyzed and 2) when EF-Tu leaves the ribosome. These 2 lags are though to be important for the 99% accuracy of protein synthesis.
2) EF-G also has a bound GTP and binds around the A-site. It helps with the conformational change and movement of the ribosome to reset the system. When GTP is hydrolyzed, it dissociates, allowing the small ribosomal subunit to move over as well to match the new location of the large ribosomal subunit.

Question 31

Explain the 2 lags that EF-Tu introduces into the system

Answer 31

1) When GTP is hydrolyzed
2) when EF-Tu leaves the ribosome.

These 2 lags are though to be important for the 99% accuracy of protein synthesis.

The reactivation of EF-Tu is catalyzed by EF-Ts (EF-1BETA/GAMMA in eukaryotes)

Question 32

List important protein factors or groups of factors that participate in protein synthesis and describe their function

Answer 32

Initiation:

1) aminoacyl-tRNA Synthetase: it adds the amino acid to the 3’ acceptor site of the tRNA. It also provides a proofreading mechanism via its editing site.
2) PROKARYOTES: Have IF-3 that binds and recruits small ribosomal subunit to shine-delgarno sequence, placing AUG in P-site.
3) PROKARYOTES: IF-2, that binds GTP and the formyl-Methionine tRNA, brings the tRNA to the AUG start site and hydrolyzes GTP.
4) EUKARYOTES: eIF2 with bound GTP and tRNA associates the tRNA with the P-site of the small ribosomal subunit AWAY from the mRNA. It is later hydrolyzed to allow binding of large ribosomal subunit.
5) EUKARYOTES: eIF4E binds to the 5’ cap of the mRNA to check for intact mRNA and recruit the small ribosomal subunit with the bound tRNA and eIF2-GTP
6) EUKARYOTES: eIF4G associates with eIF4E and plays a similar role.
7) EUKARYOTES: eIF2B catalyzes the reactivation of the eIF2.

Elongation:

1) EF-Tu binds to GTP and hydrolyzes it to be released. It ensures correct amino acid/tRNA binding.
2) EF-G also hydrolyzes GTP and assists in the movement of the ribosome and mRNA 3 nucleotides.

Termination:

1) Release factor binds and catalyzes the addition of water when it binds to the A-site. This cleaves the polypeptide from the mRNA

Question 33

Identify the specific steps in protein synthesis where GTP hydrolysis is thought to occur

Answer 33

Initiation:

1) PROKARYOTES: IF-2, that binds GTP and the formyl-Methionine tRNA, brings the tRNA to the AUG start site and hydrolyzes GTP.
2) EUKARYOTES: eIF2 with bound GTP and tRNA associates the tRNA with the P-site of the small ribosomal subunit AWAY from the mRNA. It is later hydrolyzed to allow binding of large ribosomal subunit.

Elongation:

1) EF-Tu binds to GTP and hydrolyzes it to be released. It ensures correct amino acid/tRNA binding.
2) EF-G also hydrolyzes GTP and assists in the movement of the ribosome and mRNA 3 nucleotides.

Question 34

Describe the cellular mechanisms that safeguard against the incorporation of incorrect amino acids into polypeptides and initiation at the wrong codon.

Answer 34

Many factors are associated with “proofreading”.

1) the aminoacyl-tRNA Synthetase. It has an editing site that will allow the incorrect amino acid on the tRNA to be cleaved off.
2) PROKARYOTES have IF-3 and the small ribosomal subunit that bind to the shine-delgarno sequence to ensure the correct codon. EUKARYOTES also have the small ribosomal subunit set the reading frame by scanning for the first AUG start site.
3) Anticodon-codon bind helps ensure the correct tRNA is bound to the correct codon.
4) Ef-Tu has a high affinity for a tRNA that has the correct amino acid bound to it. It is the “last check” to make sure everything is correct. It then brings the tRNA to the A-site to be incorporated. It also introduces 2 lags in the system which are though to give the protein synthesis its accuracy.

Question 35

Explain the features of GTPases that facilitate their function as irreversible molecular switches in the regulation of cellular processes.

Answer 35

They function as irreversible molecular switches because they are the rate limiting steps. In other words, when looking at eIF-2 with bound GTP and tRNA, when it is hydrolyzed to GDP, it is released from the tRNA and we have the initiation of the protein synthesis. However, eIF-2 cannot bind to another tRNA until it has been phosphorylated again back to GTP. This acts to regulate these cellular process. Also, if the cell is stressed, eIF2 can be phosphorylated on its alpha subunit and thus causing it to bind irreversibly to the eIF2B which reactivates it, causing protein synthesis to come to a halt. It can no longer be initiated! Furthermore, when one looks at EF-Tu and EF-G, they also need to be phosphorylated again after they are hydrolyzed.

Question 36

5 things that lead to the fidelity of protein synthesis

Answer 36

1) aminoacyl-tRNA synthetase must recognize the correct tRNA
2) Aminoacyl-tRNA synthetase must select the correct amino acid
3) mRNA must be fully processed (in eukaryotes) prior to translation initiation
4) The ribosome matches the mRNA codon to the tRNA anticodon. The correct anticodon forms a stronger interaction with the codon than an incorrect pairing
5) GTP hydrolysis and release of EF-Tu elongation factor provide short delays allowing the tRNA to be released from the A-site of the ribosome before an incorrect amino acid is irreversibly added into the peptide chain.

Question 37

Compare the functions of the shine-delgarno box and 7-methylguanosine cap in translation in prokaryotes and eukaryotes

Answer 37

The shine-delgarno sequence is where IF-3 and the small ribosomal subunit bind to in prokaryotes. It is located just upstream of the AUG start site, thus the small ribosomal subunit and the ribosome in general starts at the AUG start site.

The 5’ Cap of the eukaryotic mRNA is where the small ribosomal subunit with the eIF-2-GTP and tRNA bound will bind to. It then scans for the AUG start site.

Question 38

Explain why most eukaryotic mRNAs are monocistronic while bacterial mRNAs can be polycistronic

Answer 38

Eukaryotic mRNA is monocistronic because the small ribosomal subunit binds and associates with the 5’ Cap and then scans for the AUG start site. Therefore, protein synthesis begins near this 5’ end of the mRNA, recognizing the first AUG. (only exception is IRES which allow for cap-independent processes to occur)

In prokaryotes, the 5’ end has no special significance because there is no 5’ cap (mRNA is not processed). There can thus be multiple ribosome binding sites or shine-delgarno sites in the interior of the mRNA chain, each resulting in the synthesis of a different protein.

Question 39

Explain how the structure of prokaryotic and eukaryotic ribosomes differ

Answer 39

They differ in the number and size of their rRNA and protein components, however, they both have nearly the same structure and function similarly. The eukaryotic ribosome and ribosomal subunits are much bigger.

Question 40

Compare the antibiotic sensitivities of protein synthesis in prokaryotes and eukaryotes

Answer 40

There are antibiotics that work only on bacteria and those that work only on eukaryotes and others that work on both. Usually if they work only on eukaryotes they are used for research purposes or to treat some cancers.

Puromycin is an example of one that works on both

Question 41

Explain how Puromycin is an example of molecular mimicry

Answer 41

Puromycin resembles a tRNA molecule. It thus can enter the A-site of the ribosome and promote a peptide bond to bind to it. It is a dead end product though that terminates the protein synthesis because it does not have the high energy bond available.

Question 42

Protein folding: To create a functional protein, what 4 things need to occur?

Answer 42

1) Folding: needs to be appropriately folded
2) Binding to required co-factors
3) Covalent modifications
4) Assemble with any partner proteins

Protein folding takes place as the protein is being synthesized. It is important because as hydrophobic regions are being synthesized and come out of the ribosome, folding prevents them from being on the outside of the protein. When synthesis is finished, there is hopefully a fully and correctly folded protein.

Question 43

Explain the role of Heat Shock Proteins

Answer 43

Proteins can fold incorrectly with heat shock so there are heat shock proteins which are chaperones that help the protein fold. There are 2 important ones:

1) Hsp 70: It binds to the hydrophobic regions of the polypeptide chain as it is being synthesized. It uses the energy from ATP to squeeze and release the protein to hopefully cause the protein to fold correctly.
2) Hsp 60: It is completely different than Hsp 70 because it acts on the protein AFTER it has been synthesized. The exposed hydrophobic regions of the misfolded protein will interact with the hydrophobic regions right at the edge of this barrel shaped structure of Hsp 60. Inside the barrel of Hsp 60, the environment is friendly for protein folding. A GroES cap covers the Hsp 60 and then energy from ATP is used to help the protein refold. If the protein is not folded correctly, it is still released. This factor also helps prevent protein aggregates from forming.

Question 44

Explain the differences in Transcription and Translation between prokaryotes and eukaryotes

Answer 44

These differences can be exploited in the creation of antibiotics.

1) In prokaryotes, transcription and translation occur in the same compartment where in eukaryotes they are separated via a nucleus. This allows prokaryotes to transcribe and translate at the same time
2) The mRNA in prokaryotes is not processed. It is thought to be this way since it is all in the same compartment
3) In eukaryotes, the mRNA is produced in the nucleus and processed which is thought to be necessary for its transport to the cytoplasm
4) In eukaryotes there are 3 different RNA polymerases, prokaryotes only have 1
5) Prokaryotic cells are polycistronic, eukaryotes are monocistronic
6) The prokaryotic and eukaryotic ribosomes differ in size. Prokaryotes have a 30S and 50S to make a 70S. Eukaryotes have a 60S and 40S to make an 80S