Translational Regulation: General Comments

1. Translational Regulation: General Comments • Can be global (e.g., changes in energy levels can affect translation of all mRNAs), gene-specific or regulon-specific. • Rate-limiting (most regulated) step is usually initiation. • Often involves phosphorylation of initiation factors (and sometimes ribosomal proteins). • mRNAs often compete for factors or ribosomes (one consequence of this: decreasing overall translation increases competition, which can change the patterns of protein produced). • Gene or regulon-specific regulation usually involves some specialized proteins that bind to the mRNAs being regulated.

2. Regulation of Globin Translation in Reticulocytes • Reticulocytes are precursors of erythrocytes • Synthesize mainly hemoglobin (95% of protein synthesis) Hemoglobin = heme cofactor & apoproteins (a, b) reticulocytes erythrocytes Avian cells

3. Rabbit Reticulocytes are used extensively for studying translation and its regulation • Reticulocytes normally make up only a few % of blood cells • Phenylhydrazine stimulates production of reticulocytes (by destroying erythrocytes); can become up to 80% of blood cells • Very active lysates can be prepared from reticulocytes recovered from fresh blood (stores well at -160◦C) • Lysates faithfully translate mRNA, and will even respond to certain regulatory compounds like heme • Low in ribonuclease activity

4. Heme availability regulates globin translation via eIF2 • If hemeis limiting, a protein kinase (HCR, heme-controlled repressor) phosphorylates eIF2a (one of three subunits of eIF2) • Phosphorylated eIF2 binds more tightly to eIF- 2B, doesn’t release, eIF2 can’t recycle Function: prevent wasteful synthesis of globin

5. eIF2 trimer tRNAiMet = Normal cycling of eIF2 Fig. 17.33a

6. eIF2 trimer Step 6 is blocked tRNAiMet Fig. 17.33b

7. eIF2, Interferons, and Viruses • Interferons are anti-viral proteins induced by viral infection • Repress translation by triggering phosphorylation of eIF2a • Kinase is called DAI, for double-stranded-RNA- (dsRNA)-activated inhibitor of protein synthesis • dsRNA triggers the same pathway (mimics virus) Role: Block reproduction of the virus

8. The role of rRNA in Peptide Bond Formation The ribosome is a ribozyme. Chapters 18.3, 19.1

9. The Elongation Cycle (in prokaryotes) Fig. 18.10

10. Fig. 19.18

11. Antibiotics that inhibit protein synthesis by binding to ribosomes. Chloramphenicol inhibits peptidyl transferase (PT) activity! Inhibits PT on 80S cytoplasmic ribosomes Fig. 18.11 3rd ed.

12. Puromycin resembles tyrosyl-tRNA, binds to the A site, accepts peptide from peptidyl-tRNA (catalyzed by PT). Fig. 18.11

13. Fig. 18.21 Puromycin release assay for PT: (1) load the P site with labeled poly-Phe by adding poly U to a translation mix, (2) add puromycin, (3) follow puro-peptide released. 50S subunit contains the PT activity, which is blocked by the antibiotics.

14. Fig. 18.23 The fragment assay uses CAACCA-f[35S]Met, which binds to the P site, and puromycin, which binds to the A site. PT activity indicated by formation of fMet-puromycin. Ribosomes (or 50S subunits) from E. coli (E) and Thermus aquaticus (T) treated with protein destroying agents still have peptidyl transferase activity.

15. Fig. 18.25 3rd ed. 99% deproteinized 50S subunits from T. aquaticus have peptidyl transferase activity that is inhibited by antibiotics and RNase T1.

16. Composition of the E. coli ribosome 50S subunit 23S & 5S RNA + 34 proteins 30S subunit 16S RNA + 21 proteins Fig. 3.16

17. Gross anatomy of the E. coli ribosome. head platform stalk Central protuberance ridge platform stalk Fig. 19.5 3rd ed.

18. The 50S subunit with the tRNAs bound in the E,P,A sites Modeled from crystal structures of the ribosomes of Thermusthermophilus at ~8 angstroms resolution in the presence and absence of the tRNAs. Fig. 19.7 3rd ed.

19. tRNAs bound mostly to RNA! 19.1f

20. Peptidyl-tRNA interacts with the 30S subunit at the anticodon end, and with the 50S subunit at the acceptor end. Similar to Fig. 19.4