DNA Translation

Question 1

Describe the genetic code.

Answer 1

They are redundant (more than 1 codon can code for same amino acid), non-overlapping, unambigious and universal.

64 codons, 61 code for amino acids and 3 triplets for stop codons in translation.

They must be read in the correct reading frame.

Question 2

Describe the structure of tRNA.

Answer 2

A single RNA strand abount 80 nucleotides long.

When flattened into 1 plane, tRNA looks like a cloverleaf. Although its L-shaped in 3D.

Each has a specific amino acid at one end and an anticodon at the other, this pairs with complementary codons on mRNA.

Question 3

What are the 2 events needed for accurate translation?

Answer 3

Correct match between tRNA and amino acid, this is done by enzyme aminoacyl-tRNA synthetase.

Correct match between tRNA anticodon and mRNA codon.

Question 4

What is the ‘wobble’?

Answer 4

The third base of codon is flexible in pairing with anticodon, so some tRNAs can bind with more than 1 codons. This flexible pairing is called wobble.

Question 5

How are tRNAs synthesised and modified?

Answer 5

tRNAs transcribed by RNA polymerase III as precursor molecules that need to be spliced and modified.

They are covalently modified before exit from the nucleus (1/10 nt is modified in mature tRNAs; more than 50 types of modifications).

Question 6

Describe the binding sites of ribosomes.

Answer 6

A (amino acid) site - holds tRNA that carries the next amino acid to be added.

P (polypeptide) site - holds tRNA that carries the growing nucleotide chain.

E (exit) site - discharged tRNAs leave the ribosome.

Question 7

How is protein synthesis initiated?

Answer 7

A small ribosomal subunit binds to the mRNA and an initiator tRNA.

Small subunit moves along mRNA until it reaches a start codon (AUG).

Initiation factos bring a large ribosomal unit to complete translation initiation complex.

Question 8

How can bacteria translate multiple peptides from a single mRNA?

Answer 8

In prokaryotic cells there is usually a polycistronic messenger. This means that more than 1 proteins are encoded by a singular mRNA.

Consequently they have more than 1 ribosome binding sites surrounding specific initiator codons for each specific polypeptide chain to be translated.

Question 9

What happens during the elongation stage?

Answer 9

Amino acids are added one by one to the preceding amino acid.

Each addition involves proteins called elongation factors and occurs in 3 steps: codon recognition, peptide bond formation, and translocation.

Question 10

Describe the formation of the initiation complex.

Answer 10

Initiator tRNA binds with methionyl and a small ribosomal subunit. There is also an initiation factor elF2 bound to GTP.

Initiation factor tRNA is bound to the P site (not usually the case). This is the pre-complex.

The pre-comlpex shifts along the mRNA (circular structure) using ATP to reach the start codon (AUG). There are more than 1 AUG, so they are skipped until the correct one is determined (embedded in a correct context, or sequence around it).

The GTP bound to elF2 will be hydrolyzed to start initiation.

Large ribosomal subunit binds to form the initiation complex.

The 2nd tRNA (aminoacyl-tRNA) will arrive with its amino acid at the A site. This amino acid is bound to methionyl via a peptide bond.

Question 11

Describe the general process of the elongation stage.

Answer 11

The amino acid of the newly bound charged tRNA will need to make a peptide bond with the previous amino acid in the polypeptide chain.

The entire chain will need to shift over with the now bound polypeptide tRNA occupying the P-site, and the previous tRNA will be released from the E-site.

Then a newly bound charged tRNA will repeat the process and grow the polypeptide chain to eventually reach a stop codon.

Specific elongation factors needed for this process.

Question 12

Describe the role of the elongation factors in elongation.

Answer 12

EF-Tu (Elongation factor Thermo Unstable):

• Bound to GTP, therefore has high affinity for aa-tRNA.
• Forms the ternary complex.
• It brings the loaded tRNA into the A-site.
• Can also proofread by checking if hydrolyzation between anticodon and codon is correct. If not, it can aid in their dissociation.

EF-G:

• Translocates the tRNA and mRNA complex.
• Moves the whole mRNA-tRNA complex along by 1 codon. I.e. The peptidyl-tRNA from A to P and deacetylated tRNA from P to E.

Question 13

Describe the structure of the ribosome.

Answer 13

The ribosome is a ribozyme. It’s made of proteins and RNAs.

RNA is the catalytic part of the ribosome.

16S (18S in eukaryotic cells) rRNA brings together codons and anticodons and the 23S rRNA catalyzes formation of peptide bonds.

Question 14

Describe the process of termination.

Answer 14

Stop codons mark the end of translation.

Occurs when a stop codon reaches the A site
A site accepts protein called a release factor.

Release factor leads to the addition of a water molecule instead of an amino acid.

This reaction releases the polypeptide and the translation assembly comes apart.

Question 15

Describe the process of termination.

Answer 15

Stop codons mark the end of translation.

Occurs when a stop codon reaches the A site, then A site accepts the protein called release factor.

Release factor leads to the addition of a water molecule instead of an amino acid.

This reaction releases the polypeptide and the translation assembly comes apart.

Question 16

What are polyribosomes?

Answer 16

A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome).

Polyribosomes enable a cell to make many copies of a protein very quickly.

Question 17

Describe the 2 classes of release factors.

Answer 17

Class 1 binds to ribosomes in response to a stop codon in the ribosomal A site. This triggers the release of the polypeptide chain.

Eukaryotes: a single class 1 RF, eRF1 that can recognize all 3 stop codons. 
Prokaryotes: 2 types, ones that can recognize UAA and UAG (RF1) and ones that can recognize UAA and UGA (RF2).

Class 2 binds to GTP and enhances the efficiency of translation termination.

They do this by either promoting the release of class 1 RF after peptidyl release, or forming a complex with class 1 RF before the stop codon.

Question 18

How does selenoproteins vary in the genetic code?

Answer 18

Slenocysteine is coded specially by a UGA codon, usually this is a stop codon (translational recoding).

If cells are present in the absence of selenium, translation would terminate at the UAG codon, resulting in a truncated, non-functional enzyme.

Question 19

How can Upf proteins recognise abnormally spliced mRNAs?

Answer 19

If mRNA is able to pass the nuclear pore and enter the cytoplasm, a mechanism would be able to recognise and check the mRNA.

Upf proteins will recognise the presence of exon junction binding proteins and a stop codon. Exon junction binding complexes are always upstream of the right stop codon.

If an exon junction binding complex is downstream of the stop codon then Upf proteins trigger mRNA degradation.

Question 20

How can Upf proteins recognise and degrade abnormally spliced mRNAs?

Answer 20

If mRNA is able to pass the nuclear pore and enter the cytoplasm, a mechanism would be able to recognise and check the mRNA.

Upf proteins will recognise the presence of exon junction binding proteins and a stop codon. Exon junction binding complexes are always upstream of the right stop codon.

If an exon junction binding complex is downstream of the stop codon then Upf proteins trigger mRNA degradation.

Question 21

Which proteins begin to fold whilst being synthesized?

Answer 21

Nascent polypeptide chains begin to fold and sometimes immediately bind to cofactors.

Also is modified and binds to other protein subunits.

When released from ribosomes, it’s immediately ready to work.

Question 22

What are molecular chaperones?

Answer 22

Molecular chaperones are proteins that guide the folding of most proteins.

One of the first types discovered is called hsp70.

Hsp70 - heat shock protein 70 - called heat shock proteins because when they were first discovered, it was for the role of responses of cells under temperature increase.

Question 23

How do molecular chaperones work?

Answer 23

They recognize short stretches of hydrophobic amino acids during protein synthesis and attaches to help with folding.

If it doesn’t work the first time, they can reapproach the polypeptide chain and attempt again until folding is achieved.

Question 24

How do hsp70 molecule chaperones work?

Answer 24

They recognize short stretches of hydrophobic amino acids during protein synthesis and attaches to help with folding.

If it doesn’t work the first time, they can reapproach the polypeptide chain and attempt again until folding is achieved.

Question 25

Describe the hsp60 molecular chaperones work?

Answer 25

They are like a container containing incorrectly or incompletely folded proteins.

After several cycles of remodeling of the contents, the proteins are exposed to specific hydrophobic protein sites.

The structure is closed.
Proteins achieve its final structure inside and is then released.

Both hsp proteins recognizes via hydrophobic regions. Therefore they are critical as they are regions in proteins which are usually not expressed in correctly folded proteins.

Question 26

What are proteasomes?

Answer 26

Proteasome is a protease, so it’s an enzyme which degrades proteins.

Question 27

What happens when the protein refolding isn’t possible?

Answer 27

In case refolding isn’t possible, then proteins are sent to a Proteasome to be degraded.

Question 28

What are proteasomes?

Answer 28

Proteasome is a protease, so it’s an enzyme which degrades proteins.

A huge system with specific domains containing active sites which are hidden.

Question 29

What happens when the protein refolding isn’t possible?

Answer 29

In case refolding isn’t possible, then proteins are sent to a Proteasome to be degraded.

Question 30

Describe the process by which a protein travels through the proteasome to be degraded.

Answer 30

A protein needs to be degraded, this is marked by a polyubiquitin chain (protein used to indicate degradable polypeptide chain).

They enter the unfoldase ring, which has a receptor site for polyubiquitin.

After entering, the polyubiquitin chain is discarded and then recycled.

Protein enters the catalytic site of the central cylinder and is degraded there. Then it’s released through the cap.

Question 31

What are the 2 ways to induce protein degradation?

Answer 31

Ubiquitin ligase can be activated via phosphorylation or allosteric transition.

Phosphorylation of protein exposes a site of the protein which sends it to degradation.