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 heme is limiting, a protein kinase (HCR,
  heme-controlled repressor) phosphorylates eIF2a
  (one of three subunits of eIF2)
• Phosphorylated eIF2 binds more tightly to
  eIF- 2B, doesnt release, eIF2 cant 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
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.
11
Puromycin resembles tyrosyl-tRNA, binds to the A
site, accepts peptide from peptidyl-tRNA
(catalyzed by PT).
Fig. 18.11
12
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.
13
Fig. 18.23
The fragment assay uses CAACCA-f35SMet, 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.
14
Fig. 18.25 3rd ed.
99 deproteinized 50S subunits from T. aquaticus
have peptidyl transferase activity that is
inhibited by antibiotics and RNase T1.
15
Composition of the E. coli ribosome
50S subunit 23S 5S RNA 34 proteins 30S
subunit 16S RNA 21 proteins
Fig. 3.16
16
Gross anatomy of the E. coli ribosome.
head
platform
stalk
Central protuberance
ridge
platform
stalk
Fig. 19.5 3rd ed.
17
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.
18
tRNAs bound mostly to RNA!
19.1f
19
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