1. Translation The Relationship Between Genes and Proteins 13 th Week Gihan E-H Gawish, MSc, PhD Ass. Professor Molecular Genetics and Clinical Biochemistry KSU

2. Table of Contents Getting from gene to protein: genetic code Getting from gene to protein: translation  Translation Initiation Translation Initiation  Translation Elongation Translation Elongation  Translation Termination Translation Termination  References

3. From gene to protein: genetic code Central Dogma  Information travels from DNA to RNA to Protein Is there a one-to-one correspondence between DNA, RNA and Protein? –DNA and RNA each have four nucleotides that can form them; so yes, there is a one-to-one correspondence between DNA and RNA. –Proteins can be composed of a potential 20 amino acids; only four RNA nucleotides: no one-to-one correspondence. –How then does RNA direct the order and number of amino acids in a protein?

4. From gene to protein: genetic code How many bases are required for each amino acid? (4 bases) 2bases/aa = 16 amino acids—not enough (4 bases) 3bases/aa = 64 amino acid possibilities Minimum of 3 bases/aa required What is the nature of the code?  Does it have punctuation? Is it overlapping?  Crick, F.H. et al. (1961) Nature 192, 1227–32. (http://profiles.nlm.nih.gov/SC/B/C/B/J/ )http://profiles.nlm.nih.gov/SC/B/C/B/J/  3-base, nonoverlapping code that is read from a fixed point.

5. From gene to protein: genetic code Nirenberg and Matthaei: in vitro protein translation  Found that adding rRNA prolonged cell-free protein synthesis  Adding artificial RNA synthesized by polynucleotide phosphorylase (no template, UUUUUUUUU) stimulated protein synthesis more  The protein that came out of this reaction was polyphenylalanine (UUU = Phe)  Other artificial RNAs: AAA = Lys; CCC =Pro

6. From gene to protein: genetic code Nirenberg:  Triplet binding assay: add triplet RNA, ribosomes, binding factors, GTP, and radiolabeled charged tRNA (figure)figure UUU trinucleotide binds to Phe-tRNA UGU trinucleotide binds to CYS-tRNA  By fits and starts the triplet genetic code was worked out.triplet genetic code  Each three-letter “word” (codon) specifies an amino acid or directions to stop translation.  The code is redundant or degenerate: more than one way to encode an amino acid

7. From gene to protein: Translation Components required for translation:  mRNA  Ribosomes  tRNA  Aminoacyl tRNA synthetases  Initiation, elongation and termination factors Animation

8. Translation: initiation Ribosome small subunit binds to mRNA Charged tRNA anticodon forms base pairs with the mRNA codon Small subunit interacts with initiation factors and special initiator tRNA that is charged with methionine mRNA-small subunit-tRNA complex recruits the large subunit Eukaryotic and prokaryotic initiation differ slightlyEukaryoticprokaryotic Animation

9. Translation: initiation The large subunit of the ribosome contains three binding sites  Amino acyl (A site)  Peptidyl (P site)  Exit (E site) At initiation,  The tRNA fMet occupies the P site  A second, charged tRNA complementary to the next codon binds the A site.

10. Translation: elongation Elongation Ribosome translocates by three bases after peptide bond formed New charged tRNA aligns in the A site Peptide bond between amino acids in A and P sites is formedPeptide bond Ribosome translocates by three more bases The uncharged tRNA in the A site is moved to the E site.

11. Translation: elongation  EF-Tu recruits charged tRNA to A site. Requires hydrolysis of GTP  Peptidyl transferase catalyzes peptide bond formation (bond between aa and tRNA in the P site converted to peptide bond between the two amino acids)  Peptide bond formation requires RNA and may be a ribozyme-catalyzed reaction

12. Translation: termination Termination Elongation proceeds until STOP codon reached  UAA, UAG, UGA No tRNA normally exists that can form base pairing with a STOP codon; recognized by a release factor tRNA charged with last amino acid will remain at P site Release factors cleave the amino acid from the tRNA Ribosome subunits dissociate from each other Review the animation of translation