RNA translation

Question 1

Ribosome; discuss function and structure

Answer 1

megaaaa RNA-protein complex that contains the catalytic center important for peptide bond formation.

Contains two subunits (large and small)

has three binding sites: A-site, P-site, and E-site.

Small subunit has decoding groove for mRNA

large subunit has peptidyl transferase center (PTC)

Question 2

mRNA, know start and stop codons

Answer 2

Contains the nucleotide sequence that encodes the protein. 3 nucleotides form codon which transcribes 1 AA. Start: AUG Stop: UAA, UGA, UAG.

Question 3

tRNA, discuss fx and structure

Answer 3

Adaptors that “read” the mRNA and deliver correct amino acid to ribosome.

contains anti-codon that binds codon of mRNA.

tRNAs have acceptor stem that is attached to a amino acid.

“wobble” means that some tRNAs can bind multiple codons.

Question 4

Aminoacyl tRNA synthetase

Answer 4

Protein enzymes that put the right AA onto the right tRNA. Each AA/tRNA has its own unique aminoacyl tRNA synthetase.

Question 5

Initiation factors (difference bacteria, eukaryotes)

Answer 5

proteins that bring ribosome to the mRNA and assist in getting translation machinery assembled. Three in prokaryotes, over a dozen eukaryotes.

Question 6

elongation factors

Answer 6

proteins that deliver tRNAs to ribosome and help with translocation (i.e. the process of moving the ribosome down the mRNA)

Question 7

Termination/recycling factors

Answer 7

Proteins that end process at a stop codon, dissociate proteins so they can be used again.

Question 8

Degeneracy

Answer 8

There are multiple codons per AA. We have 64 codons that only encode ~20 AA. Gives ability to protect proteins w/ silent mutations.

Question 9

Frame shift mutation

Answer 9

The insertion or deletion of a base pair. Will fuck up all AA downstream of the frame shift.

Question 10

Missense Mutation

Answer 10

Change in 1 nucleotide that changes a single AA in the protein.

Question 11

Silent Mutation

Answer 11

Change in 1 nucleotide that has no effect on protein due to degeneracy.

Question 12

Nonsense Mutation

Answer 12

Change in 1 nucleotide results in stop codon. Forms truncated protein

Question 13

Sense Mutation

Answer 13

Change in 1 nucleotide results in stop codon removal. Forms overly long protein.

Question 14

Identify the important differences between bacterial and eukaryotic translation, especially in regard to initiation

Answer 14

Initiation:
Bacteria: The ribosome binds essentially right at the start codon due to the Shine-dalgarno sequence and three initiation factors work to assemble the full ribosome.

Eukaryotes: eIF4E is required to bind to the 7-methyl guanosine cap. Leads to binding of many other eIF’s and eventually binding small ribosomal subunit. The small subunit then scans down the RNA (process uses ATP) until it lands on an AUG start codon within a kozak sequence. This brings in large subunit and initiator tRNA.

Internal ribosome entry sites (IRES) are regions in the mRNA that make it so the cap is not needed to bring in the ribosome. Used commonly by viruses.

Question 15

Initiation

Answer 15

Bacteria: The ribosome binds essentially right at the start codon due to the Shine-dalgarno sequence and three initiation factors work to assemble the full ribosome.

Eukaryotes: eIF4E is required to bind to the 7-methyl guanosine cap. Leads to binding of many other eIF’s and eventually binding small ribosomal subunit. The small subunit then scans down the RNA (process uses ATP) until it lands on an AUG start codon within a kozak sequence. This brings in large subunit and initiator tRNA.

Internal ribosome entry sites (IRES) are regions in the mRNA that make it so the cap is not needed to bring in the ribosome. Used commonly by viruses.

Question 16

Elongation

Answer 16

each step results in one more amino acid added to the polypeptide chain.

Amino-acid laden tRNA enters A-site thanks to the help of EF1A (Euk)/EF-TU (Pro) w/ GTP bound

Release of EF1A and hydrolysis of its ATP form peptide bond

Translocation occurs, mRNA and tRNA move one codon over w/ hydrolysis of GTP connected to EF2.

tRNA previously in the P-site moves into the E-site, and is ejected (GTFO!)

tRNA previously in A-site is in P-site bound to the polypeptide chain. A-site is open and the cycle starts all over again.

Question 17

Termination

Answer 17

Reading of a stop codon (UAA, UAG, UGA) causes ending of elongation and dissociation of subunits. Done when a recycling factor recognizes stop codon in A-site.

Question 18

Ribosome recycling

Answer 18

After breaking off, ribosomal subunits move back to 5’ end of mRNA. Evidence mRNA is circularized to make this process more efficient. Protein binds both to cap and polyA tail.

Question 19

Cap-independent initiation

Answer 19

IRES (internal ribosome entry site), regions in mRNA that bind the ribosome obviating the need for cap binding.

Eukaryotes used this as a technique to avoid viral infection. Would shut down global translation (Shut down 4E).

Viral cells responded by making their own IRES and shutting down translation w/ a protease that degrades eIF4-G

Question 20

Interferon

Answer 20

produced by virally infected cells; will be released and communicate to other cells (paracrine signaling) to cause those cells to suppress translation in order to avoid production of viral proteins.

Question 21

mRNA editing (post transcription)

Answer 21

RNA can be edited through chemical modification (e.g. deamination) post-transcription. An example of this is changing the base in an mRNA transcript so that a stop codon is produced. This leads to proteins of different lengths.

Question 22

Rapamycin treatment

Answer 22

acts via MTOR to inhibit eukaryotic cap-dependent translation, this occurs via the upregulation of phosphorylation of 4E-BP

Rapamycin blocks mTOR blocks 4E-BP phosphorylation. Non phosphorylated 4E-BP binds to 4E, preventing formation eIF4E

Question 23

eIF2-α phosphorylation

Answer 23

eIF2-α is important in bringing the initiator tRNA in to start translation. When eIF2-α is phosphorylated, it becomes inactive. This is one way to have global shutdown of translation.

Question 24

Identify antibiotics that operate by affecting translation

Answer 24

Aminoglycosides (streptomycin), macrolides (azithromycin, erythromycin), tetracyclines (tetracycline), Other (chloramphenicol)

Question 25

Describe how intracellular levels of iron can be regulated by translation, using this as an example of how protein‐mRNA interactions regulate translation

Answer 25

Fe levels are heavily regulated b/c it is necessary for cellular processes but high levels are toxic 🕱💀☠

If Fe levels are low:
Up level of transferrin receptor (TFR) transports Fe into cell. With low levels of iron, IRE-BP binds to IRE (iron response element) protecting it from degradative enzymes. Translation of TFR goes up. With high levels of Fe, Fe binds to IRE-BP removing it from RNA. IRE is degraded and less transferrin is made.

If Fe levels are high
Ferritin binds and sequesters Fe. IRE-BP binds to IRE with low levels of Fe (IRE in 5’ UTR so this physically blocks translation). High levels of Fe, IRE-BP dissociates from RNA and translation occurs.