Translational Regulation: General Comments
description
Transcript of Translational Regulation: General Comments
![Page 1: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/1.jpg)
Translational Regulation: General Comments
1. Can be global (e.g., changes in energy levels can affect translation of all mRNAs), gene-specific
or regulon-specific.2. Rate-limiting (most regulated) step is usually
initiation.3. Often involves phosphorylation of initiation factors
(and sometimes ribosomal proteins). 4. mRNAs often compete for factors or ribosomes (one
consequence of this: decreasing overall translation increases competition, which can change the patterns of protein produced).
5. Gene or regulon-specific regulation usually involves some specialized proteins that bind to the mRNAs being regulated.
![Page 2: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/2.jpg)
Regulation of Globin Translation in Reticulocytes
• Reticulocytes are precursors of erythrocytes• Synthesize mainly hemoglobin (95% of protein
synthesis) Hemoglobin = heme cofactor & apoproteins (, )
reticulocytes erythrocytesAvian cells
![Page 3: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/3.jpg)
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
![Page 4: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/4.jpg)
Heme availability regulates globin translation via eIF2
1. If heme is limiting, a protein kinase (HCR, heme-controlled repressor) phosphorylates eIF2 (one of three subunits of eIF2)
2. Phosphorylated eIF2 binds more tightly to eIF-2B, doesn’t release, eIF2 can’t recycle
Function: prevent wasteful synthesis of globin
![Page 5: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/5.jpg)
eIF2 trimer
tRNAiMet
Fig. 17.33a
= Normal cycling of eIF2
![Page 6: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/6.jpg)
Fig. 17.33b
eIF2 trimer
tRNAiMet
Step 6 is blocked
![Page 7: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/7.jpg)
eIF2, Interferons, and Viruses
• Interferons are anti-viral proteins induced by viral infection
• Repress translation by triggering phosphorylation of eIF2
• 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
![Page 8: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/8.jpg)
The role of rRNA in Peptide Bond Formation
The ribosome is a ribozyme.
Chapters 18.3, 19.1
![Page 9: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/9.jpg)
Fig. 18.10
The Elongation Cycle (inprokaryotes)
![Page 10: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/10.jpg)
Fig. 19.18
![Page 11: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/11.jpg)
Fig. 18.11 3rd ed.
Antibiotics that inhibit protein synthesis by binding to ribosomes.
Inhibits PT on 80S cytoplasmic ribosomes
Chloramphenicol inhibits peptidyl transferase (PT) activity!
![Page 12: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/12.jpg)
Fig. 18.11
Puromycin resembles tyrosyl-tRNA, binds to the A site, accepts peptide from peptidyl-tRNA (catalyzed by PT).
![Page 13: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/13.jpg)
Fig. 18.21
50S subunit contains the PT activity, which is blocked by the antibiotics.
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.
![Page 14: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/14.jpg)
Fig. 18.23
Ribosomes (or 50S subunits) from E. coli (E) and Thermus aquaticus (T) treated with protein destroying agents still have peptidyl transferase activity.
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 offMet-puromycin.
![Page 15: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/15.jpg)
Fig. 18.25 3rd ed.
99% deproteinized 50S subunits from T. aquaticus have peptidyl transferase activity that is inhibited by antibiotics and RNase T1.
![Page 16: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/16.jpg)
Fig. 3.16
Composition of the E. coli ribosome
50S subunit 23S & 5S RNA + 34 proteins
30S subunit 16S RNA + 21 proteins
![Page 17: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/17.jpg)
Central protuberance
stalk
ridge
headstalk
platform
platform
Gross anatomy of the E. coli ribosome.
Fig. 19.5 3rd ed.
![Page 18: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/18.jpg)
The 50S subunit with the tRNAs bound in the E,P,A sites
Modeled from crystal structures of the ribosomes of Thermus thermophilus at ~8 angstroms resolution in the presence and absence of the tRNAs.
Fig. 19.7 3rd ed.
![Page 19: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/19.jpg)
19.1f
tRNAs bound mostly to RNA!
![Page 20: Translational Regulation: General Comments](https://reader035.fdocuments.us/reader035/viewer/2022062810/56815e6b550346895dccea02/html5/thumbnails/20.jpg)
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