Lecture 7 Bio 99

Ribosome

Has a small and large subunit. True in both bacteria and eukaryotes, but eukaryotes are more complex.

Large Subunit

Catalyzes peptide bond formation.

Small Subunit

binds to mRNA, tRNA assembly

rRNA processing

Transcribed as one large transcript.

Harry Noller Exp 1

Proved that ribosome is necessary for peptide bond formation.

• Add puromycin and fMet to ribosome. The puromycin stops translation, here it’s being used as substrate for peptide bonding.

• Adding denaturing compounds, none of which stopped the bonding.

• Proteins alone w/ no ribosome produced no peptide bonding.

Harry Noller Exp 2

• Treat small subunit with kethoxal (damages guanine).

• Then combine large subunit, and add to tRNA & mRNA.

• Activity goes down drastically with kethoxal, and there is almost no activity without any tRNA and mRNA (needed for translation). Which shows that the damage is being done to rRNA only.

Ribosome itself is active in translation, not just a passive structure. The ribosome is a ribozyme.

Ribosome tRNA binding sites

A, P, and E

Translation initiation basic steps

1. Small Subunit binds to mRNA

2. Small Subunit binds initiator tRNA

3. Large Subunit binds to the rest

The final product has small/large subunit, mRNA (start codon at P site), and initiator tRNA (paired with the start codon)

Shine-Dalgarno Sequence

A consensus sequence in front of the start codon.

• Physically recruits the small ribosomal subunit to the mRNA.

• Aligns the subunit so the start codon is positioned in the P-site

Bacterial Initiation Proteins

IF-1,2, & 3

IF-1

In initiation, blocks A site to prevent random tRNAs from binding.

IF-2

In initiation, uses GTP to deliver initiator tRNA to the P site, so it can pair with AUG start codon.

IF-3

Prevents large subunit from binding until the small subunit is ready. This is so the small subunit can first bind to the start site/mRNA.

Bacterial Initiation Step 1

a.) Blocking

IF-1 & IF-3 bind to the small subunit, blocking A site and keeping large subunit away.

b.) mRNA Recruitment

mRNA attaches to small subunit, uses Shine-Dalgarno sequence to position start codon at P site.

Bacterial Initiation Step 2 & 3

IF-2 (bound to GTP) binds to initiator tRNA and delivers it to P site, they bind, then the large subunit joins and GTP is hydrolyzed, which kicks off IF-1,2,3. Only initiator tRNA can bind to the rRNA. The initiator tRNA and P site can only bind with eachother.

How is eukaryotic initiation different than bacterial?

• Initiator tRNA does NOT require start codon to assemble into small subunit. (Bacteria needs to bind start codon to P site first.)

• elF5B, bound to GTP (like IF-2), joins initiator tRNA at P site.

• THEN mRNA is loaded, elF4F complex binds to the 5’ cap of mRNA (not start codon) and escorts it to the small subunit.

• The binding to the complex requires hydrolysis of ATP.

• Complex scans along mRNA until it finds start codon, then stops. (This implies that eukaryotic translation starts with first start codon from 5’ end.)

• Kozak sequence (consensus) helps identify start codon.

• 2 GTP and 1 ATP used, only 1 GTP in bacteria.

elF4F

Complex in eukaryotes, recruits ribosome.

• elF4E: Binds to 5’ cap of mRNA

• elF4A: ATPase & RNA helicase (needed because of mRNA structure, to load ribosome.)

• elF4G: Glue/Adapter, binds to elF3 and elF4E, which links mRNA to thw small subunit.

IRES

An IRES is an RNA sequence that recruits eIF4F and translation machinery to an internal site within an mRNA, allowing translation initiation without starting from the 5′ cap. Skips scanning. Can make eukaryotic polycistronic transcripts.