NOVEMBER 5, 2008 *THE RIBOSOME: This is also known as the “site.” It consists of a combination of ribosomal RNA (rRNA) and ribosomal proteins (30-50). Large and small subunits come together to form the ribosome. *STEPS OF TRANSLATION: These include 1) tRNA charging 2) initiation 3) elongation and 4) termination. tRNA charging is technically the preparatory stage for the steps of translation, but it must be done before translation can take place. We have already discussed tRNA charging, so will start now with initiation. *INITIATION: The first step is the small ribosomal subunit binding to the mRNA at the initiation site (the start codon AUG). The AUG start codon is usually several bases down from the G cap. AUG becomes the frame, and then you frame off the nucleotides that follow into codons. Here is an example of a framed fragment. Then the charged tRNA with the specific anticodon for the start site binds at the triplet in the P site of the small subunit. Following the association of the tRNA with the small subunit, the large subunit combines with the small to form the complete ribosome. *ELONGATION: This is where codons are read (3 nucleotides at a time), and a string of polypeptides are formed. Peptidyl transferase forms peptide bonds that links 2 amino acids. When met was broken off of the tRNA in the P site, the top configuration went from which allowed the tRNA bond to break free from the P site and release. Therefore the tRNA is ejected because it no longer adheres to the codon. Now the unit is going to ratchet the message through the ribosome. It moves mRNA through it and the A site becomes the new P site. There is now an open A site and the new tRNA comes in with its amino acid. More peptide bonds form, and you have a growing polypeptide sticking out a hole in the ribosome. It keeps going until a stop codon is encountered! Eventually you hit a stop codon. This stop codon signals release factors (usually some kind of small proteins). This causes a conformational change, which causes a disassociation of the tRNA, small, and large subunits. How two things can happen to the message. It can be read again, and go through the process again, or the Poly A tail may have gotten too short and the message will get chewed up and recycled. The tRNAs will go back to be charged. *EXAMPLE PROBLEM: A peptide as 100 amino acids… 1. What are the number of tRNAs needed? 100 (because there is one amino acid for each tRNA) 2. What is the number of codons present? 101 (a codon for each amino acid, plus a stop codon at the end) 3. What is the number of anticodons? 100 (anticodons are the top parts of the tRNAs so the number of anticodons equals the number of tRNAs) 4. What is the minimum number of nucleotides for translation? 304 (there are 3 nucleotides for each codon therefore 3 x 101 = 303, then there is another nucleotide from the G cap, therefore 303 + 1 = 304) 5. Would this likely be the number of nucleotides found in the transcriptional unit if this were a mammalian gene? No, because if you have a mature message, there are no introns so it’s going to be smaller than the transcript written for it. Also, caps and poly A tails change the number of nucleotides. *EXAMPLE PROBLEM: Answer the questions based on the sequence below 5’ A-U-G-C-U-G-U-A-C-G-U-G-U-U-C 1. How many codons are present? 5 (be sure to frame and see this) 2. Is a stop codon present? No (stop codons include UAA, UAG, and UGA) 3. Which is the start codon? AUG 4. How many amino acids will be added? 5 (one for each codon that is not a stop codon) 5. What is the sequence of the individual tRNA? U-A-C-G-A-C-A-T-G-C-A-C-A-A-G 6. What is the amino acid sequence? Met-Leu-Tyr-Val-Phe (you will be given a genetic dictionary if you are asked to produce the amino acid sequence) *AMINO ACIDS: Proteins are peptide chains which are made of individual amino acids strung together. This is the general structure for an amino acid: The side group R is what makes amino acids different from each other. R can range from a simple H to a very complex ring structure. The peptide bond is the bond combining two amino acids: Peptidyl transferase is the enzyme responsible for forming the peptide bond between the two animo acids. H20 is released as a product of this reaction, therefore this reaction is known as a dehydration synthesis reaction. *TEST: This will conclude what will be on Test 3. Friday’s lecture will not be included. This test will be all multiple choice. Bring pencils, but Dr. Wooten will provide scantrons. Dr. Wooten said that students did not do well on replication on the last exam, so to review DNA replication as it would probably show up again on this test.