Theme 2- Module 3 (Translation)

Question 1

Is each tRNA molecule identical? How does this aid in their function?

Answer 1

No, bc of this, they can translate a specific RNA codon into a specific amino acid

Question 2

Describe the structure of a tRNA molecule?

Answer 2

Single RNA strand, 70-90 nucleotides in length

Large degree of complementarity. Many stretches of hydrogen bonding between complementary nucleotide bases –> Four double helix segments and three loops

Folds into a roughly L-shaped 3D structure

Question 3

What is the “anticodon region” of tRNA?

Answer 3

Specific nucleotide triplet that is complementary to a specific mRNA codon that codes for a specific amino acid

Question 4

The anticodons in tRNA are written in which direction relative to the mRNA codons?

Answer 4

3’–>5’ direction but align to the mRNA codons in the 5’–>3’ dir

Question 5

What is located at the 3’ end of a tRNA molecule?

Answer 5

Protruding amino acid attachment site that is made up of a single CCA nucleotide sequence

Terminal A (adenine) = actual point of attachment for an amino acid during tRNA molec activation

Question 6

What is aminoacyl tRNA synthase?

Answer 6

Enzyme that matches/activates the tRNA with specific amino acids

Question 7

Are all aminoacyl tRNA synthases the same?

Answer 7

No, each of them is specific to the amino acid and tRNA that it will bind to

Question 8

Describe the process of tRNA activation

Answer 8

The active site of synthase enzymes recognise the antocodon end of the tRNA and the region of the amino acid attachment site

Once bound to active site, these enzymes catalyze the covalent attachment

This leads to a charged tRNA or an aminoacyl being released from the enzyme (and can now deliver amino acids)

Question 9

Does activating tRNA require energy?

Answer 9

Yes

Question 10

How many aminoacyl tRNA synthases are there?

Answer 10

20 – one for each amino acid

Question 11

True or false: mRNA can code for 64 possible codons. This means that there are 64 tRNA molecules to match each codon

Answer 11

False

There are 45 tRNA molecules which means that some are able to bind to more than one codon

Question 12

What does “wobble” mean in terms of tRNA?

Answer 12

Explains why some tRNA molecules able to bind to more than one codon

While the first base (5’ end) of the codon will bind to the last base (3’ end of anticodon), there is a greater flexibility for base pairing between the third nucleotide of the codon and the corresponding base of a tRNA anticodon

Question 13

How do prokaryotes initiate translation?

Answer 13

Initiation complex binds at Shine-Dalgarno sequences (ribosome binding sites)

(prok don’t have a 5’ cap)

Question 14

How do eukaryotes initiate translation?

Answer 14

Translation initiation complex forms towards 5’ cap of mRNA and scans it until an AUG codon is encountered

Question 15

Where are Shine-Dalgarno sequences usually located on the mRNA?

Answer 15

A few bases upstream of AUG

Question 16

Why is the mRNA in prokaryotes polycistronic (able to code for more than one peptide with the same mRNA strand)?

Answer 16

Shine-Dalgarno sequences can occur multiple times and translation can happen at all of them simultaneously.

Prok have functionally related genes grouped together along its DNA; transcribed together

Question 17

The initiation of translation req the assembly of various components, such as….

Answer 17

• Two ribosomal subunits
• mRNA (to be translated)
• charged tRNA methionine (anticodon for AUG)
• initiation factors

Question 18

Describe the assembly of the translation initiation complex in eukaryotes

Answer 18

Initiation factors bind to 5’ cap

Recruits small ribosomal subunit

Other initiation factors bind to charged tRNA that has methionine

This complex moves along mRNA until an AUG is encountered. When it finds the AUG, the large ribosomal subunit binds and so does the next charged tRNA molecule

Question 19

Where does the large ribosomal subunit get the energy to bind to the rest of the initiation complex?

Answer 19

GTP hydrolysis

Question 20

When the partially assembled initiation complex is moving along, trying to find the start codon, which direction does it move in?

Answer 20

5’ to 3’

Question 21

Once the the initiation complex is assembled, what happens to the initiation factors?

Answer 21

They are released

Question 22

True or false: all amino acids are synthesized from the amino end to the carboxyl end

Answer 22

True

Question 23

Which amino acid is the first one to be found at the amino end of the polypeptide?

Answer 23

Methionine

Question 24

Which site of the ribosome do all incoming charged tRNA molecules enter and bind to prior to each amino acid being incorporated into growing polypeptide chain?

Answer 24

Aminoacyl

Question 25

What are the three sites of the ribosomal subunit (in order from entrance to exit)?

Answer 25

Aminoacyl
Peptidyl
Exit

Question 26

Which site will methionine be located in at the start of translation (unlike all of the other amino acids)?

Answer 26

Peptidyl

Question 27

How are incoming charged tRNA molecules able to enter and bind to the sites on the ribosome (considering the amino acid hasn’t even been added yet)?

Answer 27

Seq of mRNA coding is read by the ribosome in successive, nonoverlapping groups of three nucleotides

Question 28

Each charged tRNA is delivered to the ribosome with a…?

Answer 28

GTP-bound elongation factor

Question 29

What happens to the GTP-bound elongation factor as the tRNA enters the A-site on the ribosome?

Answer 29

When correct codon-anticodon pairing is made, GTP turns to GDP and the aminoacyl end of tRNA is released from the elongation factor

Question 30

What is the max number of charged tRNA molecules that can be present within the ribosome at any given time?

Answer 30

2

The E site does not have any charged tRNA

Question 31

Following the binding of a charged aminoacyl tRNA, what happens?

Answer 31

There is a conformational change in the ribosomal RNA that allows for a peptidyl-transferase reaction to occur between the amino acids to form a polypeptide chain

Question 32

Describe the peptidyl transferase reaction that occurs between the amino acid from the P-site and the amino acid from the A-site

Answer 32

Forms a condensation rxn as a peptide bond, and a transfer of the growing polypeptide chain onto the tRNA that is in the A-site

Question 33

What allows the ribosome to continue to translocate along the length of the mRNA molecule?

Answer 33

GTP-bound elongation factors cause the deacylated tRNA to move from the P-site to the E-site. When the next tRNA enters the A-site, the deacylated tRNA is released from the E-site

Question 34

What happens once the ribosome reaches the STOP codon?

Answer 34

GTP-bound release factors bind to the A-site and catalyse the hydrolysis of the bond between the terminal amino-acid in the polypeptide and the tRNA in the P-site

Further GTP hydrolysis enables dissociation of translation complex

Question 35

Describe the “one-gene-one-enzyme” hypothesis that was established by George Beadle and Edward Tatum

Answer 35

Based on the fact that Neurospora crassa can grow on a minimal medium (that contains only simple sugars, etc.).

Hypo: must have some enzymes produced by a specific gene that convert these simple substances into the amino acids and vitamins that are needed for growth

Short form: each gene contains the info needed to make each enzyme

Question 36

How do they know that neurospora are able to synthesize their own arginine (amino acid)?

Answer 36

Bc they are able to grow well in a medium that lacks arginine, therefore, they must be able to make it themselves

Question 37

What is the metabolic pathway for argenine synthesis?

Answer 37

Precursor
Ornithine
Citrulline
Arginine

Question 38

In argenine synthesis, the transition between steps requires what?

Answer 38

A separate enzyme for each step

Question 39

Describe the procedure of Srb and Horowitz’ experiment that tested the one-gene-one-enzyme hypo

Answer 39

Performed a genetic screen of radiation treated neurospora to det whether there were specific genes that produced each of the three enzymes that are needed in argenine synthesis

Raised these colonies on a medium that was supplemented with either nothing (precursor), ornithine, citrulline, or arginine (to prevent mutation)

Question 40

What were the results of Srb and Horowitz’ experiment?

Answer 40

Medium with argenine —> growth
Other mediums –> no growth

In the mediums with ornithine and citrulline, there was an inhibition of growth

Question 41

Why was the surface of the liquid in the test tube in Srb and Horowitz’ experiment slanted?

Answer 41

To allow room for growth

Question 42

What conclusions were made from Srb and Horowitz’ experiment?

Answer 42

Radiation produced mutations in the genes that encode for the necessary enzymes for the production of argenine

Found three mutants: arg1, arg2, arg3. Arg1 cells lack enzyme needed to make precursor into ornithine. Arg 2 lack enzymes needed to make ornithine into citrulline and so on…

Question 43

Why is the one-gene-one-enzyme hypo usually called the “one gene, one polypeptide” hypo now?

Answer 43

Bc they found out later that genes dictate structure of all proteins

Question 44

What is the human proteome?

Answer 44

Rep the full number of proteins that are expressed by all of the hereditary info in our DNA (aka our genome)

Question 45

There are over 1 million proteins in our bodies, but only 20-25000 genes that encode for them. What does this mean?

Answer 45

More than one protein can be produced from a single gene

Question 46

A mutation in arg2 prevents argenine synthesis. The defect can be rescued by the addition of which molecule?

Answer 46

Citrulline