Translation (Theme 2: Module 3)

Question 1

What happens during translation?

and end result is what type of structure?

Answer 1

cellular components are able to read the genetic message in the mRNA sequence and translate this message into the specific primary amino acid sequence of a protein

end result: 3 dimensional

Question 2

What is the role of tRNA in translation?

Answer 2

enabling the translation of info into the mRNA genetic message to a polypeptide

Question 3

tRNA molecules are able to transfer:

Answer 3

amino acids to a growing polypeptide strand in a ribosome (specific manner as each type of tRNA molecule is not identical - specific mRNA codon to a specific amino acid)

Question 4

tRNA molecule is made up of:

Answer 4

a single RNA strand ranging between 70-90 nucleotides in length

-has a large degree of complementarity which results in many stretches of hydrogen bonding between complementary nucleotide

Question 5

shape of tRNA (theres 2):

Answer 5

clover leaf, 4-double helical segments and characteristic loops

or L-shaped: whole tRNA molecule can fold upon itself (3D)

Question 6

anticodon region of tRNA

Answer 6

specific nucleotide triplet that forms complementary base pairs w/a specific mRNA codon that codes for a specific amino acid

-written in a 3’ - 5’ direction and align properly w/mRNA codons in the 5’ - 3’ direction

Question 7

actual point of attachment for an amino acid during tRNA molecule activation

Answer 7

Terminal A/ adenine

Question 8

3’ end of tRNA

Answer 8

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

Question 9

aminoacyl tRNA synthase

Answer 9

carries out the activation of a tRNA molecule w/a specific amino acid (each is specific to the type of tRNA and corresponding amino acid that it will bind to)

Question 10

How does aminoacyl tRNA synthase activate tRNA molecules

Answer 10

• active site of these enzymes recognize the anticodon end of the tRNA and the region of the amino acid attachment site (leads to formation of 20 aminoacyl tRNA synthetases, one for each amino acid)

-once bound to active site, enzymes can then catalyze the covalent attachment of a tRNA molecule to its amino acid using the energy from ATP hydrolysis

-leads to a charged tRNA molecule, aminoacyl tRNA, being released from the enzyme which can now deliver its specific amino acids to a growing polypeptide chain on a ribosome

Question 11

Primary sequence of amino acids in the translated polypeptide occurs when:

Answer 11

there is correct pairing of the tRNA anticodon with the appropriate mRNA codon.

(base pairing between a codon in mRNA and an anticodon in tRNA)

Question 12

Wobble?

Answer 12

explains the redundancy of the genetic code

due to the chemical nature of codon-anticodon pairing interactions, the first base (5’) of the codon will bind w/the last base (3’ of anticodon), there is a greater flexibility for base pairing between the third nucleotide of a codon and the corresponding base of a tRNA anticodon.

Question 13

Process of translation roughly:

Answer 13

-AUG codon in mRNA codes for the amino acid methionine which signals to the protein translation machinery to begin translating the mRNA at that location

(instead of 64, there are 45 tRNA molecules, meaning that some may be able to bind to more than one codon, this explained by wobble)

-as an mRNA molecule is shifted through a ribosome, specific mRNA codons are translated into amino acids one-by-one
-these amino acids are attached one-by-one to a growing polypeptide chain until translation in terminated

Question 14

location of translation in prokaryotes vs in eukaryotes:

Answer 14

prokaryotes: assembly + translation in cytoplasm, happens immediately after mRNA is transcribed from the RNA template

eukaryotes: cytoplasm (transcription in nucleus), two diff processes

Question 15

1. Initiation of translation in prokaryotes vs in eukaryotes

Answer 15

eukaryotes: occurs when a translation initiation complex forms towards the 5’ cap of the mRNA and then scans the mRNA until an AUG start codon is encountered

prokaryotes: have no 5’ caps, translation initiation complex will assemble at one or more ribosome binding sites called Shine-Dalgamo sequences (located upstream of start AUG codon)

Question 16

Reading frames of prokaryotes

Answer 16

have specific reading frames for more than one protein along a single mRNA strand. Translation of this type of polycistronic mRNA in prokaryotes can occur because prokaryotes can have functionally related genes grouped together along the prokaryotic DNA and these genes are often transcribed as a single unit from one promoter

-every gene has 3 reading frames, 1 will result in the functional protein we created

Question 17

2. assembly of initiation process (both prokaryotes and eukaryotes):

Answer 17

large + small subunits of ribosomes will assemble to form a functional ribosome only when attached to an mRNA molecule

-requires the assembly of: 2 ribosomal subunits, mRNA that requires translation, charged tRNA methionine and initiation factors (help assemble initiation complex)

Question 18

Eukaryotic Translation

Answer 18

-initiation factors will bind to the 5’ cap of mRNA (allows for the recruitment of a small ribosomal subunit)

-at the same time, other initiation factors will bind to the tRNA that is charged w/methionine

-this partially assembled initiation complex will move along mRNA in a 5’ to 3’ direction until an AUG (start codon) is encountered

-once this all occurs, the large ribosomal subunit is able to bind to the rest of the initiation complex using the energy released from GTP hydrolysis

-Finally, the next charged molecule can join the ribosome

Question 19

3. Charged tRNA is delivered to the functional ribosome

Answer 19

-initiation factors are released once ribosomal translation complex is completely assembled

-polypeptides are synthesized from amino acid to carboxyl end

-methionine (located at P, peptidyl, site of ribosome) will be the first amino acid that is found at the amino acid end of a polypeptide

-each subsequent charged tRNA enters and binds within the aminoacyl (A) site of the large ribosomal subunit

-accomplished as the sequence of mRNA coding for amino acids is read by the ribosome in successive, non overlapping groups of 3 nucleotides

-each incoming charged tRNA is delivered in association with a GTP-bound elongation factor, when the correct codon-anticodon pairing has been made, the GTP is then hydrolysed and the aminoacyl end of the tRNA is released from the elongation factor

Question 20

Peptide Bond formation (allowed by translation)

Answer 20

occurs following binding of a charged aminoacyl tRNA, there is conformational change that is induced in the rRNA that allows for peptidyl
(transferase rxn to occur -involves formation of condensation rxn as a peptide bond, and the transfer of the growing polypeptide chain onto the tRNA that is in the A-site)

• ribosome will continue to translocate along the length of the mRNA molecule
  (enabled by the binding of the GTP-bound elongation factors that cause the deacylated tRNA to move from the P-site to the exit (E-site)

-the subsequent aminoacyl -tRNA to enter the A-site will then allow for the release of the deacylated tRNA from the E site

Question 21

4. Termination of Translation

Answer 21

occurs at: stop codon

-once stop codon is reached on mRNA sequence, GTP-bound release factors will bind to the A-site and catalyze the hydrolysis of the bond between the terminal amino acid in the polypeptide and the tRNA in the P-site

-further GTP hydrolysis will also enable the dissociation of the transcription complex, including the ribosomal subunits and any remaining bound tRNA

Question 22

George Beadle and Edward Tatum

Answer 22

established the relationship between genes and proteins. The “one-gene-one-enzyme hypothesis”

-based on the fact that Neurospora can grow well on minimal growth medium, as a result, neurospora must have enzymes produced by a specific gene that convert these simple substances into the amino acids and vitamins that are needed for growth

Question 23

Adrian Srb and Norman Horwitz

Answer 23

further tested “one-gene-one-enzyme” hypothesis

-performed a genetic screen of radiation treated neurospora to determine whether there are specific genes that produced each of the 3 enzymes that are needed in the metabolic cascade of arginine synthesis…

-were aware that treating the cells w/radiation would lead to potential mutations in the bread mold DNA so they decided to conduct the genetic screen by raising colonies of radiation treated cells on a medium that was supplemented by nothing else (did not have orthinine, citrulline, orginine added)

They observed that when growing the radiation treated Neurospora on medium that was supplemented w/arginine, there was continual growth of the fungus (there exists a positive control that indicated that w/supplemented arginine, the fungus was still able to undergo growth)

However, when cells were placed in a non-supplemental medium, there was no growth (led to belief that radiation must have produced mutations in the genes that encode for the necessary enzymes for the production of arginine by the Neurospora cells)

when examining cells that were placed in ornithine or citrullinr-only supplemented medium, it was found that there was also inhibition of growth

Question 24

Results of “one-gene-one-enzyme”

Answer 24

3 mutants had been discovered: arg 1, arg 2, arg 3 representing mutant neurospora that had mutations for enzyme 1, 2, and 3 respectively that are needed for the production of arginine

This experiment convinced researchers of the accuracy of this hypothesis, and further research identified that genes do not only code for enzymes in an organism, but rather that genes dictate the structures of all proteins, each produced by a specific gene product

as a result, referred to as “one-gene-one-polypeptide” hypothesis

Question 25

Exception to “one-gene-one-protein” hypothesis

Answer 25

Human genome had 20-25,000 protein encoding genes, provided evidence that more than one protein can possibly be produced from a single gene

there is added complexity that exists from genome to proteome. Specifically, post-translational modifications of proteins that are translated from the same DNA allows for production of diverse proteins that will have very specific roles