1. Translation is the RNA- directed synthesis of a polypeptide In the process of translation, a cell “reads” a genetic message and builds a polypeptide accordingly. Polypeptide Ribosome Trp Phe tRNA with amino acid attached Amino acids tRNA Anticodon Codons UUUUGGGGC A C C C AAA C C G 5 3 mRNA

2. Molecular Components tRNA  The function of tRNA is to transfer amino acids from the cytoplasm pool of amino acids to a growing polypeptide in a ribosome.  A tRNA molecule translates a given mRNA codon into a certain amino acid.  This is possible because a tRNA bears a specific amino acid sequence at one end, while at the other end is a nucleotide triplet that can base-pair with the complementary codon on mRNA (anti-codon).

3. tRNA Structure  A tRNA consists of a single RNA strand (about 80 nucleotides).  This single strand can fold back on itself and create a 3- dimensional structure.  Flattened on a plane: cloverleaf shape.  3-D shape: roughly L-shaped.  A loop extending from one end contains the anti-codon.  The 3’ end serves as the attachment site for an amino acid. Amino acid attachment site Hydrogen bonds Anticodon

4. tRNA Production  tRNA molecules are transcribed from DNA.  Occurs in nucleus of eukaryotes. After transcription, tRNA leaves nucleus.  Occurs in cytoplasm of prokaryotes.  The correct matching up of tRNA and amino acid is carried out by enzymes called aminoacyl-tRNA synthetases.  The active site of each type of aminoacyl-tRNA synthetase fits only a specific combination of amino acid and tRNA.  About 20 different synthesases.  These enzymes require energy from the hydrolysis of ATP.

5. Aminoacyl-tRNA synthetase (enzyme) Amino acid PPP Adenosine ATP P P P P P Adenosine tRNA Adenosine P tRNA AMP Computer model Amino acid Aminoacyl-tRNA synthetase Aminoacyl tRNA (“charged tRNA”)

6. Numbers of tRNA  There are only about 45 different tRNA molecules.  Some tRNAs must be able to bind to more than one codon!  tRNAs are versatile!  Such versatility is possible because the rules for base pairing between the third nucleotide base of a codon and the corresponding base of the tRNA anticodon are relaxed.  Wobble!  Wobble explains why the synonymous codons for a given amino acid more often differ in their third nucleotide base.

7. Ribosome Basics  Ribosomes consist of a large subunit and a small subunit, each made up of proteins and one or more ribosomal RNAs (rRNA).  The ribosome is only functional when these subunits join together and attach to a mRNA molecule.  Ribosomes are constructed in the nucleolus.  Ribosomal protein translated in cytoplasm.  rRNA transcribed in nucleus.  Ribosomal protein imported from cytoplasm, and then assembly occurs.  One-third of the ribosomes mass is rRNA.  Three rRNA molecules in bacterial ribosomes.  Four rRNA molecules in eukaryotic ribosomes.

8. Ribosome Anatomy  P site: holds the tRNA carrying the growing polypeptide chain.  A site: holds the tRNA carrying the next amino acid t be added to the chain.  E site: where discharged tRNAs leave the nucleus.  Exit tunnel: area that growing polypeptide passes through.

9. Ribosome Association and Initiation of Translation  Initiation of translation brings together mRNA, a tRNA bearing the first amino acid of the polypeptide (methionine), and the two ribosomal subunits.  In eukaryotes…  Small subunit, with the initiator tRNA already bound, binds to the 5’ cap of the mRNA.  The small subunit scans downstream along the mRNA unit it reaches the start codon (AUG).  Finding the start codon established the correct codon reading frame.  The union of mRNA, initiator tRNA, and small ribosomal subunit is followed by the attachment of a large ribosomal subunit, completing the translation initiation complex.  Initiation factors bring these components together.  Cell expends energy obtained by hydrolysis of GTP.  The initiator tRNA then sites in the P site of the ribosome.  Polypeptide then is synthesized in the N-terminus to C-terminus direction.

10. Initiator tRNA mRNA 5 5 3 Start codon Small ribosomal subunit mRNA binding site 3 Translation initiation complex 5 3 3 U U A A G C P P site i  GTPGDP Met Large ribosomal subunit EA 5 Ribosome Association and Initiation

11. Elongation of the Polypeptide Chain  In the elongation stage, amino acids are added one by one to the previous amino acid at the C-terminus of the growing chain.  Elongation factors aid in the addition of amino acids to the growing polypeptide chain.  Energy is expended in first and third step.  Codon recognition requires hydrolysis of one molecule of GTP, which increases accuracy and efficiency of this step.  One more GTP is hydrolyzed in the translocation step.  The ribosome moves in the 5’  3’ direction along the mRNA.

12. Amino end of polypeptide mRNA 5 E A site 3 E GTP GDP  P i P A E P A GTP GDP  P i P A E Ribosome ready for next aminoacyl tRNA P site Elongation

13. Termination of Translation  Elongation continues until a stop codon in the mRNA reaches the A site of the ribosome.  A release factor, a protein shaped like an aminoacyl tRNA, binds directly to the stop codon in the A site.  The release factor causes the addition of a water molecule.  This reaction breaks the bond between the complete polypeptide and the tRNA in the P site.  The polypeptide is then released through the exit tunnel.  The breakdown of the translation assembly then occurs, which requires the hydrolysis of two more GTP molecules.

14. Release factor Stop codon (UAG, UAA, or UGA) 3 5 3 5 Free polypeptide 2 GTP 5 3 2 GDP  2 i P Termination

15. Completing and Targeting the Functional Protein Protein Folding and Post- translational Modifications Targeting Polypeptides to Specific Locations  During synthesis, polypeptide begins to fold spontaneously.  Due to primary structure.  Chaperons assist with folding.  Post-translational Modifications:  Amino acids may be chemically modified.  Enzymes may remove one or two amino acids from leading end.  Polypeptide chain may be cleaved.  Polypeptide synthesis always begins at a free ribosome.  Polypeptides bound for excretion or the endomembrane system signal for the ribosome to attach to ER.  Signal peptide is recognized by signal-recognition particle.  This particle escorts the ribosome to the ER.  Proteins are either inserted into the ER or embedded in the ER membrane.

16. Ribosome mRNA Signal peptide SRP 1 SRP receptor protein Translocation complex ER LUMEN 2 3 4 5 6 Signal peptide removed CYTOSOL Protein ER membrane

17. Making Multiple Polypeptides  In both bacteria and eukaryotes multiple ribosomes translate an mRNA at the same time.  Once a ribosome is fare enough past the start codon, a second ribosome can attach to the mRNA.  Polyribosome!

18. Concept 17.5: Mutations of one or a few nucleotides can affect protein structure and function  Mutations are changes in the genetic material of a cell or virus.  Point mutations are chemical changes in just one base pair of a gene.  The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein.

19. Types of Small-Scale Mutations  Point mutations within a gene can be divided into two general categories:  Nucleotide-pair substitutions  One or more nucleotide-pair insertions or deletions

20. Substitutions  A nucleotide-pair substitution replaces one nucleotide and its partner with another pair of nucleotides.  Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code.  Missense mutations still code for an amino acid, but not the correct amino acid.  Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein.

21. Insertions and Deletions  Insertions and deletions are additions or losses of nucleotide pairs in a gene.  These mutations have a disastrous effect on the resulting protein more often than substitutions do.  May cause a premature stop.  May cause extensive missense.  Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation.

22. Wild type DNA template strand mRNA 5 5 3 Protein Amino end A instead of G (a) Nucleotide-pair substitution 3 3 5 MetLysPheGly Stop Carboxyl end TTTTT TTTTTAAAAA AAAACC C C A AAAAA GGGG G CC GGGUUUUUG (b) Nucleotide-pair insertion or deletion Extra A 3 5 5 3 Extra U 53 TTT T TT TT A AAA A A T GGGG G AAA AC CCCCA T 35 53 5 T TTTTAAAACCA A CC T TTT T A AAAA TGGG G U instead of C Stop UAAAAA GGGU UUU UG Met Lys PheGly Silent (no effect on amino acid sequence) T instead of C TTTTT AAAACCA GT C T A T TTAAAACCA G CC A instead of G CA AAAA GAGUUUUUG U AAAA GG GUUU G A C AA UU AA UUGU G GC UA G A U AUA A UGUGUU C G MetLys Phe Ser Stop Met Lys missing Frameshift causing immediate nonsense (1 nucleotide-pair insertion) Frameshift causing extensive missense (1 nucleotide-pair deletion) missing TTTT T T CAA C C A A CG A GTTTAAA A A TGG G C LeuAla Missense A instead of T T TTT TAAA AA C GG A G A CA U AAA G G G UUUUU G T TTTTA T AAA C GG G G Met Nonsense Stop U instead of A 3 5 3 5 5 3 3 5 5 3 3 53 MetPheGly No frameshift, but one amino acid missing (3 nucleotide-pair deletion) missing 3 5 5 3 5 3 U TC A A A C A T TACG TA G T T T GG AA T C T T C A A G Met 3 T A Stop 3 5 5 3 5 3 Figure 17.24