Supplemental Instruction
IowaStateUniversity / Leader: / Matt C.
Course: / Biol/Gen 313
Instructor: / Dr. Myers & Dr. Vollbrecht
Introduction: This note sheet summarizes the molecular components and steps in protein translation.Also, since we’ve almost entirely focused on prokaryotic translation, this is what we will focus on here as well.
Concepts
- Initiation.
- Elongation.
- Termination.
Material
- Initiation.
Initiation is probably the most complex of these processes. We’ll try and break this down into 4 steps. IF-1, IF-2, IF-3, GTP, the 30S ribosomal subunit, the 50S ribosomal subunit, fMet-tRNAfMet, and the mRNA transcript are the molecules involved in initiation.
Step 1:In the first step, you start building your ribosomal complex. The 30S subunit will bind to the mRNA transcript by recognizing the Shine-Dalgarno sequence. The 16S rRNA which is part of the 30S subunit is what recognizes the Shine-Dalgarno sequence. IF-3 will also bind at this step. IF-3 keeps the 50S subunit from attaching too early. If the 50S docks prematurely, fMet-tRNAfMet won’t be able to enter the P site. It also helps the tRNA bind.
Step 2:Next, IF-1, IF-2-GTP, and fMet-tRNAfMet all bind. IF-1 you don’t have to know too particularly, but IF-1 helps prevent early docking of charged tRNAs to the A site. IF-2 will associate with GTP to form a complex and become activated. When IF-2 is activated it can bind to both the newly forming ribosomal complex and fMet-tRNAfMet. The tRNA will bind to the start codon downstream from the Shine-Dalgarno sequence. The codon site where this tRNA attaches will eventually be the P site when the 50S binds.
Also, the fMet-tRNAfMet is the nomenclature used to describe a charged tRNA. The first part fMet-tRNAfMet indicates which amino acid the tRNA should bind to. The superscript indicates that it is charged: fMet-tRNAfMet.
Step 3: The book has this as one step, but just to really differentiate things, we’ll do it in two. What happens next is IF-2 recognizes that the tRNA, mRNA, and 30S are attached properly. After this is achieved, IF-2 releases the energy bound up in GTP by hydrolyzing GTP to GDP. Hydrolyzing GTP causes IF-2 to change its shape, releasing itself from the tRNA and ribosome and also knocking off IF-1 and IF-3. So IF-1, IF-2-GDP, and IF-3 all leave the complex and leave the mRNA, 30S subunit, and charged tRNA behind.
Step 4:The last step consists of the 50S subunit finally attaching to the ribosome complex. The fMet-tRNAfMet ends up in the P site, which is the middle site of the final ribosome. The complex is now ready to enter the elongation cycle.
- Elongation.
Elongation is the middle step, but occurs as a cycle. So after one walk-through of elongation, the ribosomal complex is ready for another round of elongation. Elongation we’ll break into 3 steps. The components involved are the ribosomal complex with its growing peptide chaine, EF-Tu, EF-Ts, EF-G, two GTPs, and a new charged tRNA.
Step 1:The first step of elongation is extremely similar to the second step in initiation. What happens is EF-Tu-GTP binds to the new charged tRNA and chaperones it to the A site – the entry point into the ribosome.
Step 2:Like IF-2, EF-Tu recognizes that the new charged tRNA has successfully entered and bound in the A site. After that, EF-Tu hydrolyzes its GTP to GDP and disassociates. After EF-Tu is out of the way, the ribosome can create the new peptide bond. The 23S rRNA performs the catalytic action to make the new bond. The 23S is a part of the 50S subunit. When the peptide bond is formed, the peptide chain is released from the old tRNA in the P-site and switches over to being held by the new tRNA in the A-site.
Step 3:This step resets all of the elongation parts to allow the cycle to run again. First, EF-Ts recharges EF-Tu with GTP. This reactivates EF-Tu and prepares it for chaperoning a new charged tRNA. Second, EF-G uses the energy provided by hydrolyzing GTP to GDP to move the ribosomal complex along 1 codon. So the ribosomal sites move 3 nucleotides along the mRNA in a 5’ to 3’ direction. This moves the old tRNA in the P-site to the E-site, where it exits the ribosome. The tRNA in the A-site that has the peptide chain bound to it now moves over into the P-site. Moving along the mRNA is called translocation.
- Termination.
Termination is the last step in protein translation and only occurs when a stop codon enters the position below the A-site. The molecules involved are the ribosomal complex with polypeptide chain, RF-1, RF-2, RF-3, and GTP.
Step 1:The 3 stop codons (UAA, UAG, and UGA) are recognized by 2 RFs (release factors). RF-1 recognizes UAA and UAG. RF-2 recognizes UAA and UGA. Both RF-1 and RF-2 are proteins; they are not tRNAs. However, they can still enter the A-site and bind to their respective codons. RF-3-GTP will bind to the protein complex after either RF-1 or RF-2 has attached.
Step 2:After RF-3-GTP binds to the ribosomal complex and verifies that one of the RFs is bound correctly, it hydrolyzes its GTP to GDP. The energy released is used to break apart all of the components of the ribosomal complex. So both of the RFs break off, the ribosomal subunits disassociate, the tRNA detaches from the mRNA and the polypeptide chain is released from the other end of the tRNA.