Protein Synthesis

Question 1

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

Answer 1

eukaryotes- different synthetase for each amino acid prokaryotes- one synthetase couples more than one amino acid.

Question 2

What are the steps in translation?

Answer 2

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

Question 3

What are tRNAs synthesized from?

Answer 3

From the four common ribonucleosides- adenosine, guanine, cytidine, uridine. After synthesis some are chemically modified to dihydrouridine (D), inosine (I), Thymine (T), and pseudouridine

Question 4

Describe the structure of tRNA

Answer 4

cloverleaf consisting of four stems stabilized by base pairing (in double helical regions) and three loops. There is also the L structure. 3’ end of the tRNA is the acceptor stem where the amino acid is attached

Question 5

Describe base pairing of the tRNA anticodon with the mRNA codon

Answer 5

Proceeds from the 5’ end of the codon. Once the first two positions are paired the exact base pairing of the wobble position is less critical.

Question 6

What is the significance of the wobble position?

Answer 6

Allows some tRNAs to recognize more than one mRNA codon making it possible for less than 64 tRNAs to recognize all 64 codons. This is why the genetic code is degenrate.

Question 7

What wobble bases match to which anticodon bases in Eukaryotes?

Answer 7

U- A,G, or I C- G or I A- U G- C

Question 8

Describe the activation of tRNA.

Answer 8

Two step activation. ATP hydrolysis is used to attach amino acid to tRNA. 1) amino acid’s carboxyl group is linked to phosphate residue in AMP. hydrolysis of pyrophosphate is energetically favorable and permits this reaction to occur. 2) Amino acid is transferred to the OH group at the 3’ end of tRNA by a ester bond

Question 9

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

Answer 9

Question 10

Which two adaptors translate the genetic code?

Question 11

What structural features of tRNA contribute to fidelity of protein synthesis?

Answer 11

1) Specific positions on the tRNA are reconized by aminoacyl-tRNA synthetase. there are special recognition sites that are recognized.
2) The aceptor stem is specific to an amino acid and the anti codon region are important in the specificity of the aminoacylation reaction as well as correct protein addition in protein synthesis.
3) The anticodon is directly recognized by the synthetase through 3 adjacent binding pockets that are complimentary to the shape and charge of the nucleotide in the anticodon.

Question 12

Explain the editing process of t-RNA aminoacyl synthetase.

Answer 12

1) The correct amino acid has a high affinity for the synthesizing site of its specific synthetase. This site will prevent larger amino acids entering. Smaller and closely related amino acids will get through though.
2) Hydrolitic editing: After the amino acid has been covalently linked to the AMP, the tRNA binds to the synthetase and tries to push the amino acid into the editing pocket. The editing pocket excludes the correct a.a. but will allow the (incorrect) closely related amino acids through. Once inside the editing site the the a.a. is hydrolyzed from the AMP or from the tRNA (if the aminoacyl-tRNA bond has already formed). The oncorrect a.a. is released.

Question 13

What is the accuracy of tRNA synthetase?

Answer 13

one mistake in 40,000 couplings

Question 14

What process is hydrolytic editing by tRNA synthetase analagous to?

Answer 14

exonucelotydic proof reading by DNA polymerase.

Question 15

What provides fidelity in protein synthesis?

Answer 15

1) Amino acyl tRNA must recognize the correct RNA.
2) Aminoacyl tRNA synthetase must select the correct amino acid
3) mRNA must be fully processed (in eukaryotes) prior to translation initiaiton
4) The ribosome mathces the mRNA codon to the correct 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 before an incorrect amino acidis irreversibly added to the peptide chain

Question 16

Ribosomes

Answer 16

coordinate protein synthesis in all cells. molecular motor that travels down the mRNA one codon at a time. At each codon it recruits an aminoacyl-tRNA and catalyzes the formation of a peptide bond.

Question 17

Selection of the correct reading frame in Prokaryotes

Answer 17

Purine rich sequence called the Shine-Delgarno sequence is -10 from the AUG or GUG codon. It is complimentary to the 3’ end of the 16S rRNA in the 30S subunit. Ribosome is positioned in the correct reading frame by base pairing between the 16S rRNA and SD sequence in mRNA (3-9 BP complimentarity)

Question 18

What sequences on the mRNA function as internal ribosomal entry sites (IRES) in prokaryotes

Answer 18

Shine-Delgarno sequence helps align the AUG in the P site, and it base pairs with a sequence near the 3’ end of the 16S rRNA.

Question 19

Initiation of protein synthesis in prokaryotes

Answer 19

1) 30S ribosomal subunit binds IF-3 (preventing premature association to 50S) the 16S rRNA of 30S binds to mRNA at the SD site next to the initiation codon and positions the initiation codon on the ribosome.
2) Ternary complex between IF-2+GTP, and fMet-initiator t-RNA binds in the P site of the 30S. Permits base pairing between initiator codon in mRNA and anti-codon in fMet-tRNA.
3) 30s and the ternary complex formed binds to 50S. Hydrolysis of the GTP on IF-2 occurs. Dissassociation of GDP, inorganic phosphate, IF-2, IF-3

Question 20

Describe the functions of the small and large ribosomal subunits

Answer 20

Small ribosomal subunit provides the framework to accurately match the mRNA codon with the tRNA anticodon. The large subunit catalyzes the peptide bond formation.

Question 21

What allows prokaryotic mRNA to be polycistronic?

Answer 21

Assembly of the ribosome on the start codon, multiple shine delgarno sequences in one mRNA.

Question 22

Initiation of protein synthesis in Eukaryotes

Answer 22

Initiation tRNA+eIF-2+GTP is loaded onto 40S with the help of eIF-3.

eIF-4G and eIF-4E bind to 5’ cap and poly-A tail of mRNA ensuring it is fully processed.

40S binds to mRNA by recognizing the cap and eIFs. Uses ATP to scan for the first AUG.

eIF-2 GTP is hydrolyzed, eIFs dissassociate, 60S binds to 40S, initiation tRNA is in the P site.

Question 23

How is eIF-2 reactivated?

Answer 23

it is reactivated by guanine exchange factor eIF2B which catalizes the reaction:

eIF-2+GDP + GTP -> eIF-2+GTP + GDP

Under cellular stress eIF2B can be shut down

Question 24

List the 5 steps of translation initiation in eukaryotes

Answer 24

Initiation of translation occurs at the first AUG codon in the majority of mRNAs

1) eIF-4 binds to the 5’ end of mRNA
2) eIF4B stimulates helicase activity of eIF4A
3) recruitment of the small ribomsomal subunit
4) Scanning for the first AUG
5) Assembly of the 80S ribosome

Question 25

Describe the 1st step of eukaryotic translation initiation

Answer 25

eIF4F (eIF4A, eIF-4E, eIF4G) bind to the 5’ cap of the mRNA aiding in recruitment of the 40s subunit (which is bound to eIF-2GTP Met-tRNA)

Question 26

What is the rate limiting step in translation?

Answer 26

The binding of eIF-4F

Question 27

What are the two main functions of the eIF4 factors?

Answer 27

1) to confirm that mRNA is appropriately processed.
2) to promote binding of the 40S subunit to the mRNA (along with eIF3).

Question 28

List the components of eIF4F and their functions

Answer 28

1) eIF4E - Cap binding protein
2) eIF4A - ATP dependent RNA helicase that unwinds the RNA secondary structure
3) eIF4G - Adapter molecule that binds bith eIF4F and eIF3

Question 29

Describe the 2nd step of eukarytic translation initiation

Answer 29

RNA secondary structure in the 5’ UTR (especially adjacent to the cap) prevents binding/scanning of the ribosome. eIF4B stimulates helicase activity of eIF4A unwinding the RNA structure.

Question 30

Describe the 3rd step of eukaryotic translation initiation

Answer 30

eIF-2•GTP binds to Met-tRNA which together bind to 40S rRNA. Then eIF3•other eIFS binds to the smal ribosomal subunit and interacts with eIF4G bringing 40S to the 5’ end of mRNA.

Question 31

Describe the 4th step in eukaryotic translatin initiation

Answer 31

The small ribosomal subunit scans for the first AUG. initiation occurs 90% of the time at the 1st AUG codon.

Question 32

Describe the 5th step of eukaryotic translation initiaiton

Answer 32

40s finds the first AUG, the 60s subunit binds, GTP hysrolysis and a conformational change occur as the final steps prior to 80S ribosome assembly. Initiation tRNA is in the P site of the ribosome.

Question 33

Describe the steps of elongation

Answer 33

Ribosome has 4 sites, A,P,E.

Initiation tRNA is in the P site, a tRNA enters the A site and base pairs with the mRNA codon. A peptide bond is formed between the amino acids in the A and P site by peptidyl trandferase in the large ribosomal subunit.

When the peptide bond is formed a conformtional change occurs that shifts the 2 tRNAs into the E and P sites. The next confomration change moves the mRNA 3 nucleotides so that the ribosome is reset and ready for the next tRNA.

The next tRNA moves to the A site and the tRNA in the E site exits.

Question 34

What is the role of elongation factors in polypetide elgonation

Answer 34

EF-Tu binds to a charged tRNA as it enters the ribsome. TP hydrolysis and exit of EF-Tu cause two short lags that provide an opportunity for an incorrectly paired tRNA to exit the ribosome prior to incorporation into the polypeptide chain (99.9% of accuracy of ribosome translation)

EFG•GTP binds near the A site and accelerates movement of the tRNAs to the P and E sites. GTP hydrolysis catalyzes the confomrational change needed to move tRNA and mRNA forward by one codon.

Question 35

Termination of translation

Answer 35

Binding of a release factor to the A site that has a stop codon forces peptidyl transferase off of the ribosome catalyzing the addition of a water molecule instead of an amino acid. This frees the carboxy terminus from the tRNA. The completed polypeptide is released and in a series of reactions that require additional proteins and GTP hydrolysis, the ribosome dissassociates into 2 seperate subunits.

Question 36

Differences in transcription and translation between prokaryotes and eukaryotes

Answer 36

Prokaryotes- mRNA is not processed with a 5’cap and poly A tail. Trancription and translation can occur simultaneously (no seperation), Polycistronic mRNA, ribosome is 50S and 30S. multiple ribosome binding sites at the SD sequence. Multiple SD sequences in the RNA each leading to a different protein.

Eukaryotes- mRNA is capped and poly adenylated, transcription occurs in the nucleus and translation in the cytoplasm, moncystronic mRNA, Ribosome is 60S and 40S. protein synthesis begins at a start codon near 5’ end of mRNA.

Question 37

Similarities between transcription and translation in eukaryotes and prokaryotes.

Answer 37

both mRNAs are synthesized with a triphosphte group at the 5’ end

Same DNA bases used, ribosomes have nearly the same strucutre and function.

Question 38

Give an example of molecular mimicry

Answer 38

puromycin mimics tRNA. There is no high energy ester linkage to add an amino acid with so causes the release of nascent polypeptide chains by its addition to the growing end. (bacteria and eukaryotes) This terminates translation.

Question 39

Steps in the creation of a functional protein

Answer 39

folding

binding required cofactors

covalent modifications

assembly with any partner proteins

Question 40

Function of hsp70

Answer 40

recognizes a small stretch of hydrophobic amino acids on the protein’s surface. with the help of Hsp40 proteins, ATP-bound Hsp70 grasp their target protein and hydrolyze ATP to ADP, undergoing conformational change that cause Hsp70 molecules to associate more tightly with the target. After Hsp40 disassociates the rapid rebinding of ATP induces the disassociation of Hsp70 protein after ADP releas, repeated cycles of Hsp binding and releasing will help to protein to refold.

Question 41

Function of Hsp60

Answer 41

A misfolded protein is captured at the edge of the barrel by hydrophobic interactions. Binding of ATP and a protein cap increases the diameter of the barrel rim, which will partially unfold the misfolded protein. Hsp60 keeps the protein in a favorable and enclosed environment allowing it to fold. After 15 seconds ATP hydrolysis occurs weakening the complex, subsequent binding of ATP ejects the protein (folded or not) and the cycle repeats. If the protein hasn’t refolded in the end it will be degraded. Hsp60 proteins are upregulated when a cell experiences heat shock.