Translation (written lesson) Q: How are proteins (amino acid chains) made from the information in mRNA? A: Translation Ribosomes translate mRNA into protein Translation has 3 steps also! 1. Translation Initiation: mRNA binds to a ribosome and a tRNA binds to the ribosome, bringing in the first amino acid (a.a.) 2. Translation Elongation: Ribosome covalently links a.a.'s together as additional tRNA's bring them to the ribosome in response to the message of the mRNA. 3. Translation Termination: When the "stop codon" of the mRNA gets to the ribosome, translation stops. mRNA is released from the ribosome; tRNA is released; newly synthesized protein is released. How does the mRNA sequence of nucleotides direct a ribosome to connect the proper protein sequence of amino acids??? The genetic code = the way that the 4 bases of RNA encode the amino acid sequence of protein. Proteins are made of monomers called amino acids. There are 20 different amino acids. Each protein is made of a different combination of a.a.'s. A messenger RNA is actually a code language that tells the ribosomes which amino acid to add first, second, third, etc. in the protein chain. The "code words" of mRNA are called codons. 3 nucleotides specify one amino acid = a codon *AUG does code for an amino acid, Methianine, therefore "Met" is always the first amino acid in a protein. What is tRNA and what is its role in translation? tRNA's carry amino acids (a.a.'s) to the ribosome during translation; the ribosome then links the a.a.'s together into a peptide chain. tRNA is a single-stranded RNA molecule the folds on itself through H- bonds between internally complementary nucleotides. Once it has folded, a tRNA is shaped like a cloverleaf. There are many different tRNA's. They are all similar to one another in their cloverleaf shape, but each one carries a different a.a. Each tRNA has an "anti-codon" that is complementary to a specific codon of mRNA. The a.a. that a tRNA carries corresponds to the codon that its own anti-codon recognizes. e.g. A tRNA whose anti-codon is AAG will carry the a.a. “Phenylalanine/Phe” because AAG is complementary to the codon UUC, which is a codon for Phe (see genetic code chart above.) GAC CUG CUG GAC 5’ 3’ AMINO ACID = NH2 AMINO ACID = ASPARTATE LEUCINE What is the structure of the ribosome? How does a ribosome build proteins? The intact ribosome is actually a combination of two subunits. The two subunits are named the "small subunit" and the "large subunit" and they must come together before translation can begin. Ribosome subunits are made of 1) ribosomal RNA (rRNA) and 2) many different proteins. Together, the rRNA's and the proteins of a ribosome's subunits act as a huge enzyme complex that can create proteins by forming new peptide bonds between a.a.'s. Every intact ribosome has three important active sites: 1) mRNA-binding site, 2) tRNA-binding site #1 (also called the "P" site) 3) another tRNA- binding site, #2 (also called the "A" site.) How does the ribosome work with the tRNA's and with the mRNA to elongate a chain of amino acids? Please see cartoon!!! 1. mRNA binds to the small subunit of a ribosome, then the 1st transfer RNA (always carries INITIATION methionine) binds to the #1 binding site of the Of TRANSLATION ribosome and to mRNA's start codon 2. large subunit joins the complex 3. A second tRNA binds to the ribosome's #2 site and to the second codon of the mRNA. (The second tRNA is chosen by its anti-codon (because its anti- codon is complementary to the codon of the mRNA.)) Initiation of Translation: Amino Acids t-RNA’s m-RNA START CODON 5’ 3’ Initiation Small Subunit Complex of Ribosome Large Subunit of Ribosome P Site A Site The ribosome catalyzes a reaction: the two amino acids carried in by the two tRNA's are linked by a peptide bond. 4. The 1st tRNA is released, but the amino acid that it carried is now covalently linked to the second tRNA's amino acid. 5. The ribosome moves tRNA #2 over into ELONGATION Of tRNA-binding site #1. This leaves TRANSLATION tRNA-binding site #2 open for a third tRNA to bind. The shift of tRNA #2 also pulls the mRNA further into the ribosome. 6. tRNA's continue to deliver a.a.'s to the ribosome, and the ribosome continues to covalently link the amino acids by forming peptide bonds between them. Each time an amino acid is added to the growing chain, the ribosome shifts the tRNA that was in binding site #2 over into site #1, pulling the mRNA in a little and exposing site #2. Elongation Phase of Translation: Amino Acids t-RNA’s 5’ 3’ New Protein NH2 TERMINATION 7. When a stop codon is reached, the newly Of TRANSLATION formed protein chain is released by the ribosome. The last tRNA is released by the ribosome. The two subunits of the ribosome fall apart. The mRNA is released by the ribosome. Amino Acids t-RNA’s 5’ 3’ New Protein STOP CODON NH2 Amino Acids t-RNA’s m-RNA COOH 5’ 3’ New Protein NH2 End of Translation Lesson REPLICATION (written lesson with still pictures) How is the double helix/DNA replicated (during the S phase of interphase)? The two strands of the parent DNA are antiparallel. This means that the 5’ end of one strand pairs with the 3’ end of the opposing strand. Adenine Hydrogen bonds to Thymine and Guanine H-Bonds to Cytosine. Thus we say that A and T are complementary and G and C are complementary. Steps in DNA replication (synthesis): 1. Parental (double-stranded) DNA molecule unwinds and hydrogen bonds between complementary pairs are broken leaving two separated, complementary backbones. The enzyme that performs the unwinding and separation is called DNA Helicase. This enzyme gradually "unzips" the two strands of the parent molecule. 5’ A T 3’ G C A T T A G C DNA Helicase Enzyme C G A T A T T A T A G C 3’ 5’ G C 2. Once the two parent strands have been separated by Helicase, new complementary strands are made, using the single- stranded parent strands as templates. The enzyme that performs the synthesis of new, complementary strands is DNA Polymerase. (Creates a polymer from monomers) 5’ A T 3’ G C DNA Polymerase A T Enzymes T A G C C G A T T A A T A T C 3’ G 5’ C G Limitations of DNA Polymerase: 1) DNA Polymerase can only add free complementary nucleotides to parental strand's 3' end. Since the strands of DNA are anti-parallel, when Helicase opens the molecule one single-stranded parent has a free 3' end, but the other has a free 5' end. Only the parent whose 3' end is single-stranded will be copied continuously by DNA Polymerase. This strand is called the Leading Strand. DNA Polymerase simply binds to the open 3' end and adds complementary bases along toward the branch point. The other parent strand must be copied in short, discontinuous pieces; therefore, it is called the lagging strand. DNA polymerase "waits" for helicase to unzip an entire segment of the double helix, it binds to the lagging strand at the branch point, which is its 3' end, and adds nucleotides away from the branch point. This creates a short fragment of daughter strand. Then the enzyme waits for another segment of lagging strand to be exposed by Helicase, and then it binds near the branch point and adds nucleotides down toward the small fragment it previously created. 5’ T 3’ A On the “Leading Strand” G DNA Polymerase C continuously adds new A T T nucleotides, as it T A follows DNA Helicase A G C C C G G On the “Lagging Strand” A T A T DNA Polymerase 5’ A T runs in the opposite A T direction of the Helicase, T A T A and therefore must make T A a series of discontinuous T A pieces (or fragments). C G C G 5’ 3’ G C 5’ 3’ G C 5’ 5 A T 3’ A T G C G C A T A T T A T A G C C C G G A T 5’ A T A T A T T A T A T A T A C G C G 3’ 5’ 3’ G C 5 G C 2) The second limitation of DNA Polymerase is that it cannot form a covalent bond between the fragments it has created on the lagging strand. The enzyme that does connect the fragments is called DNA Ligase. 5’ 5 A T 3’ A T 3’ G C G C A T A T DNA Ligase enzyme T A can connect the two T A fragments by forming G C G C a covalent bond C G between them. 3’ C G A T 5’ A T A T A T T A T A A T A T G C G C 5’ 3’ G C 5 3’ G C Since the two strands of the parent molecule were complementary, and since the two new strands are complementary to the parent, the two, double-stranded daughter molecules are identical to each other and to the original/parent DNA molecule. They are called sister chromatids. 5’ 5’ A T 3’ A T 3’ G C G C A T A T We now have two T A identical daughter T A molecules of DNA, G C G C and the cell is ready for C G a Mitosis or a Meiosis C G division.