Current LectureMCB 250 1st Edition Lecture 18Outline of Current Lecture 1. RRNA Processing in Eukaryotes2. Translation in Prokaryotes3. 30s Initiation Complex4. Large Subunit5. Fidelity6. Translocation 7. Termination8. Prokaryotes9. EukaryotesCurrent Lecture1. RRNA processing in eukaryotes- There are many rRNA genes in eukaryotes (100s) found in clusters of genes. One copy repeated after another- Transcribed by pol I. - Operons have different promoters (pol I promoters)- Transcripts are made and then must be processed to release ribosomal proteins- They are not capped or spliced- They are not capped because all machinery from mRNA loads on c terminal RNA pol II. Ifnot RNA pol II, the machinery will not load and not see the transcript coming out of thepolymerase. That is the pol II specific phenomenon- Separate polymerase pol I- Nucleolus: dark spot in nucleus where are the ribosomes are transcribed2. Translation in Prokaryotes- Initiation: base pairing with the 16s rRNA to the ribosome binding site of the message- Initiation codon and factors. - Initiator tRNA- To position the start codon of the message in the P site is done by the ribosome binding site in the 16s rRNAThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- It is initiated with a modified methionine with a formal group. It is a special tRNA. Has a slightly different sequence that is recognized and modified when the normal methioninewas on there.3. 30s Initiation complex- Role of initiation factor 3 is that I binds to the E site and prevents the interaction between the two subunits of the ribosome until it is ready- Initiation factor 1 and 2 complex with one another. 2 is bound to GTP and they bind in the A site. This complex interacts with mRNA via base pairing in the 16s rRNA and positions the start codon in the P site and initiation factor 2 recognizes the fMET tRNA and puts it in the P site and now it base pairs with the AUG start codon- fMET tRNA is different than normal met tRNA= allows it to be recognized by initiation factor 24. Large Subunit- Once that is assembled initiation factor 3 goes away. Now the large subunit of the ribosome can bind and complete the complex- There is a region in the large region of the ribosome called the Factor Binding Center. - Large subunit will bind and initiation factor 2 with GTP will find itself appropriately position in the Factor Binding Center. This will allow it to know that it is in the right position. This also triggers GTP hydrolysis and change conformation of the protein and initiation factor 1 and 2 will go away. Left with 70s Initiation complex (small subunit, large subunit, initiator codon at P site, mRNA)- Ready to make protein and enter the elongation phase now- Next tRNA with the appropriate amino acid will come to the next codon at the A site. The Amino group from A will attack the bond and the peptide bond will transfer from P site to A site- New tRNA will come in bound to its codon. Now the amino group will attack the carbon and create new peptide bond, releasing old peptidyl tRNA- Energy bond from the charged tRNA. 5. Fidelity- Ribosome has relatively good fidelity- Put in a wrong amino acid 1/1000 amino acids- How big is the average protein: 250 amino acids- Most of the time a protein wont have a problem- Ribosome only cares about the codon anticodon is base paired appropriately!- How does it know that it got the right tRNA?o Codon/anticodono Hydrogen bonds in the minor groove of the codon-anticodon with the 16s RNA. Minor grove of first and second base. IF its not posititioned right, there will be nominor grooveo EFTU –GTP has to be appropriately positioned in the Factor binding center. If its not right, the position wont be right. o Accommodation of the movement of the tRNA to carry out the reaction- Carry out reaction and the peptide is bound to the tRNA in the a site. Now you need to translocate the ribosome one codon down- EFG6. Translocation- EFG- Charged tRNA bound to EFTU mimics the EF-G shape- During elongation, the top of the A site tRNA is already moved over to allow the peptide bond to form and the peptide to go out through the channel. Now the bottom needs to move so the EF-G-GTP will come in and the GTP will hydrolyze and that will change its conformation and extends itself so that it pushes the bottom of the tRNA over and then the A site will be empty. The Ef-G will then fall off, leaving the A site empty.- A site is accessible7. Termination- There are two release factors that mimic the TRNA. It is all protein that will interact and make H bonds with the stop codons. It will hydrolyze the peptide away from the tRNA in the P site and let the protein go. Then RF-3 comes in with a GDP and removes RF 1 or 2 from the A site. - Then the ribosome release factor goes into the A site mimicking a tRNA and then a EFG-GTP comes in and translocate the RRF to the P site and everything will fall out and IF3 comes back into the E site so that the subunits do not come back together. Everything is recycled- TRANSLATION: 3 ATP’S PER AMINO ACID ADDED8. Prokaryotes- Can be polycistronic: can make more than 1 protein for a given messenger RNA- Own reading frame will have its own shine delgarno and ribosome reading site9. Eukaryotes- Always more protein involved- Ribosomes are larger- Initiation is different: eukaryotes mRNA do NOT have RBS. The Cap is recognized instead and regular methionine is used to