8.12.16 Lecture

Question 1

What is the genetic code?

Answer 1

A set of 64 codons (triplet of nucleotides) that each code for one amino acid (some amino acids have multiple codons)

Question 2

What does it mean to say that the genetic code is degenerate?

Answer 2

Most amino acids are represented by more than one codon

Question 3

Degenerate codons tend to contain the same nucleotides in the ___ positions and vary in the ___ positions.

Answer 3

First and second; third (Wobble)

Question 4

What is translation?

Answer 4

The process of translating mRNA into protein

Question 5

True or false - the genetic code varies for different organisms.

Answer 5

False - the genetic code is universal in all organisms.

Question 6

An mRNA consisting of contiguous triplet codons can be read in three different ___. How many are correct?

Answer 6

Frames; only one.

Question 7

What are the 4 basic steps of translation?

Answer 7

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

Question 8

Each ___ is specific for an amino acid.

Answer 8

tRNA

Question 9

What synthesizes tRNA?

Answer 9

RNA Polymerase III

Question 10

What is the anticodon?

Answer 10

A sequence of three nucleotides in the tRNA that base pairs with a codon in mRNA

Question 11

tRNA is modified after synthesis; why?

Answer 11

Helps maintain its folding

Question 12

Once the first two positions are paired, the exact pairing of the third position is less critical. This is the ___ position. What is the purpose of this?

Answer 12

Wobble; permits some tRNAs to recognize more than 1 codon.

Question 13

For each wobble base codon, what are the possible anticodon bases? (U, C, A, G)

Answer 13

U -> A, G, I C -> G, I A -> U G -> C

Question 14

If referring to the anticodon, the Wobble position is the ___ base. If referring to the codon, the Wobble position is the ___ base.

Answer 14

First; third.

Question 15

What catalyzes the two-step activation of a t-RNA?

Answer 15

Aminoacyl-tRNA synthetase

Question 16

Draw the two-step activation of a t-RNA.

Answer 16

Question 17

The genetic code is translated by means of two adaptors that act one after another. What are these and what do they do?

Answer 17

1. Aminoacyl-tRNA synthetase - couples the amino acid to the corresponding tRNA
2. tRNA - its anticodon base pairs with a codon on the mRNA

Question 18

How does the aminoacyl tRNA synthetase recognize the correct tRNA?

Answer 18

Extensive structural and chemical complimentarity; three adjacent binding pockets in the synthetase match the shape and charge of the nucleotides in the anticodon.

Question 19

How does the aminoactyl tRNA synthetase select the correct amino acid?

Answer 19

Proofreading activity via hydrolytic editing - an incorrect amino acid fits into the editing site and is cleaved. A correct amino acid does not fit, confirming that it is correct. The correct aminio acid has the highest affinity for the synthesis site (versus the editing site).

Question 20

The proofreading activity of aminoacyl-tRNA synthetase is very accurate; there is one mistake in ___ couplings.

Answer 20

40,000

Question 21

Describe the process by which prokaryotes select the correct reading frame for protein synthesis.

Answer 21

Shine-Dalgarno sequences are located ~10 nucleotides upstream of the initiator codon in mRNA. These sequences are complementary to the 3’ end of the 16S-ribosomal RNA in the prokaryotic 30S subunit. The ribosome is positioned for translation initiation in the correct reading frame by base pairing between the 16S rRNA and Shine-Dalgarno sequences.

Question 22

What are Shine-Dalgarno sequences?

Answer 22

Purine-rich sequences of 3-9 bases that bind the ribosome in prokaryotes in order to set the correct reading frame for translation.

Question 23

How do eukaryotes select the correct reading frame?

Answer 23

eIF2 sets the frame; AUG start codon

Question 24

What are the steps to initiation of protein synthesis in prokaryotes?

Answer 24

1. The 30S subunit base pairs with the SD sequence upstream of the AUG start codon. This positions the initiating codon in the P site of the ribosome. 30S binds IF-3, preventing premature assembly with 50S.
2. A ternary complex between IF-2, GTP, and fMet-initiator tRNA binds in the P site of 30S, permitting base-pairing between mRNA initiation codon and fMet-tRNA (methionine-formyl methionine) anticodon.
3. The large complex formed in step 2 binds 50S subunit. GTP hydrolysis occurs and GDP, inorganic phosphate, IF-2, and IF-3 dissociate.

Question 25

What do the subunits of the ribosome do?

Answer 25

The small ribosomal subunit accurately matches the codon and anti-codon. The large ribosomal subunit catalyzes peptide bond formation.

Question 26

What are the steps to initiation of protein synthesis in eukaryotes?

Answer 26

1. Initiator tRNA (with bound eIF2-GTP) is loaded into the small ribosomal subunit at the P site. 2. eIF4E and eIF4G bind to the 5' cap and the polyA tail of the mRNA, ensuring the mRNA is not broken. This aids recruitment of the 40S subunit (which has eIF2-GTP\*met-tRNA), which binds to the mRNA at the 5' end. 3. eIF-4A and 4B facilitate the unwinding of the mRNA secondary structure so 40S can scan the untranslated region until a suitable AUG is reached. ATP hydrolysis catalyzes this step. 4. eIF2-GTP is hydrolyzed, eIFs dissociate, and the large 60S ribosomal subunit binds to the 40S. Synthesis is ready to begin.

Question 27

What is the rate limiting step of translation initiation in eukaryotes and why?

Answer 27

The cleavage of eIF2-GTP to GDP; eIF2-GDP must be recycled to eIF2-GTP (catalyzed by eIF2B)

Question 28

What are the steps to elongation in protein synthesis in eukaryotes?

Answer 28

1. Ribosomes contain 4 binding sites - mRNA site, A-site, P-site, and E-site. At the end of initiation, the initiation tRNA is in the P-site. An aminoacyl-tRNA molecule binds to a vacant A-site. 2. A new peptide bond is formed between the amino acids in A and P. 3. Large subunit translocates relative to the small subunit, leaving the 2 tRNA in hybrid sites (large in P, small in A, and large in E, small in P). This is catalyzed in part by the peptidyl transferase activity of the large subunit. 4. Small subunit translocates, carrying the mRNA 3 nucleotides and resetting the ribosome

Question 29

In what direction does translation occur?

Answer 29

5' to 3'; N-term end of the protein is created first

Question 30

Elongation requires what two things?

Answer 30

GTP hydrolysis and the participation of 3 elongation factors, including 2 GTP-binding proteins

Question 31

What are the two major elongation factors in prokaryotes? In eukaryotes?

Answer 31

Prokaryotes: EF-Tu and EF-G Eukaryotes: EF-1 and EF-2

Question 32

What do EF-Tu and EF-G do?

Answer 32

1. EF-Tu\*GTP binds to a charged tRNA as it enters the ribosome. GTP hydrolysis and the exit of EF-Tu cause 2 short lags that provide an opportunity for an incorrect tRNA to exit prior to incorporation. 2. EF-G\*GTP binds near the A-site and accelerates the movement of tRNAs to the P and E sites. GTP hydrolysis catalyzes conformational changes needed to move tRNA and and advance the mRNA.

Question 33

What catalyzes reactivation of Ef-TU?

Answer 33

EF-Ts

Question 34

What are the steps of translation termination?

Answer 34

1. Stop codon is reached. 2. Release factor eRF1 binds to the ribosome's A site and forces peptidyl transferase to catalyze addition of water instead of an amino acid. 3. Frees carboxyl terminus; protein released 4. Ribosome dissociates into 2 separate units

Question 35

Why can eRF1 bind to the A site?

Answer 35

It mimics tRNA structurally and chemically

Question 36

What 5 steps contribute to the fidelity of protein synthesis?

Answer 36

1. Aminoacyl tRNA synthetase must recognize 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 with the tRNA anticodon. 5. GTP hydrolysis and release of EF-Tu elongation factor provide short lags the allow incorrect tRNA to be released before irreversible addition.

Question 37

Compare and contrast the site of translation in prokaryotes and eukaryotes.

Answer 37

Prokaryotes: Transcription and translation are coupled; protein synthesis can begin before mRNA synthesis is done (no processing) Eukaryotes: pre-mRNA is processed in the nucleus, mature message exported to the cytoplasm where translation occurs

Question 38

Prokaryotes are polycistronic; eukaryotes are monocistronic. What does this mean?

Answer 38

Prokaryotes have multiple SD ribosome binding sites and each can synthesize a different protein at the same time. Eukaryotes can only synthesize one protein at a time.

Question 39

Compare and contrast the ribosome structures of prokaryotes and eukaryotes.

Answer 39

Prokaryotes: 70S (50S - large, 30S - small) Eukaryotes: 80S (60S - large, 40S - small)

Question 40

Compare and contrast prokaryotic and eukaryotic sensitivity to antibiotics.

Answer 40

Inhibitors can act only on eukaryotes, on both eukaryotes and prokaryotes, or on prokaryotes only.

Question 41

What is puromycin?

Answer 41

An antibiotic that causes premature release of nascent polypeptide chains by its addition to the growing chain end; acts on both prokaryotes and eukaryotes. It is also an example of molecular mimicry - it looks like tRNA and halts protein synthesis by binding to the ribosome.

Question 42

What are the 4 steps required to create a functional protein?

Answer 42

1. Folding 2. Cofactor binding 3. Covalent modifications 4. Assemble with partner proteins

Question 43

True or false - proteins can begin to fold as they are synthesized.

Answer 43

True

Question 44

Which end of the protein folds first?

Answer 44

N-terminal domain

Question 45

Some proteins requires assistance from chaperones to fold. What are two families of chaperones and what do they do?

Answer 45

1. Hsp70 family: act early, recognize small stretches of hydrophobic amino acids on the surface of a protein; ATP-bound Hsp70 grabs the taret and hydrolyzes ATP to ADP. Conformational changes occur that cause tighter association of hsp70. Repeats this cycle to help protein to fold. 2. Hsp60 family: forms barrel to isolate misfolded proteins, prevent aggregation, and provide a favorable environment to refold. Captures the protein via hydrophobic amino acids on the surface.