Download Initiation in eukaryotes

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Generalized G protein cycle
(GEF)
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Make a list of translation factors that are G-proteins, or GEFs
initiation
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elongation
termination
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Energy requirements for translation
Step
Initiation
Event
Energy
amino acid activation
1 ATP
…inactive 80S ribosome…dissociation mRNA binding to 40S subunit
Elongation
Termination
-
assembly of 40S and 60S subunits
-
binding of aminoacyl-tRNA
1 GTP (EF-Tu)
peptide bond formation
Exergonic
translocation
1 GTP (EF-G)
1 GTP (RF-3)
1 ATP + 3 GTP / cycle
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‫‪TRANSLATION II‬‬
‫שיעור אחרון בביולוגיה מולקולארית‬
‫החל מהשיעור הבא נלמד שיטות יישומיות ‪-‬‬
‫כלומר הנדסה גנטית‬
‫‪8‬‬
Model of eukaryotic ribosome
• rRNAs are believed to play a catalytic role in protein
synthesis.
• After removal of 95% of the ribosomal proteins, the
60S subunit can catalyze formation of peptide bonds.
• Ribosomal proteins are now believed to help fold the
rRNAs properly and to position the tRNAs.
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Figure 6-39a Molecular Biology of the Cell (© Garland Science 2008)
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INITIATION IN
EUKARYOTES
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‫באאוקריוטים‪ ,‬רק ה‪( met-tRNA-‬האיניציאטור) נקשר לתת היח' הקטנה‬
‫של הריבוזום ביחד עם ‪.(eIF( Eukaryotic initiation factors‬‬
‫‪ tRNA‬מוטענים אחרים נקשרים ישר לאתר ה‪....P-‬‬
‫‪15‬‬
‫בהמשך‪ ,‬תת היחידה הקטנה נקשרת לקצה ה‪( '5-‬ל‪)CAP-‬‬
‫של ה‪ MRNA -‬ולפקטורי תרגום נוספים‬
‫‪16‬‬
‫תת היחידה הקטנה עם כל הקומפלקס‪ ,‬נעה בחיפוש אחר‬
‫ה‪ AUG-‬הראשון‪.‬‬
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‫פקטורים נוספים‪ ,‬המשמשים כהליקאזות נקשרים אף הם‪,‬‬
‫ומאפשרים את תנועת הריבוזום ע"ג ה‪ . MRNA-‬לאחר מציאת‬
‫ה‪ ,AUG-‬הפקטורים נופלים‪ ,‬ומאפשרים את קישור תת היח'‬
‫הגדולה = סוף האיניציאציה ותחילת האלונגציה‬
‫‪18‬‬
‫נתחיל עם‬
‫‪19‬‬
‫‪EIF2‬‬
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Figure 7-107 Molecular Biology of the Cell (© Garland Science 2008)
‫ופקטורים נוספים‬
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5’ end (cap) dependent initiation:
• The first step is the recognition of the 5’ cap by eIF4F,
which consists of three proteins, eIF4E, eIF4G and
eIF4A.
• Cap binding protein, eIF4E, binds to cap
• The N-terminus of eIF4G binds eIF4E and the Cterminus binds eIF4A
• The 40S subunit binds to eIF4G via eIF3
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Closed loop model:
• The 5’ end dependent initiation is stimulated by the
poly(A) binding protein Pabp1p, which interacts with eIF4G
• This interaction circularizes the mRNA and facilitates
formation of the initiation complex
• Mechanism to ensure that only intact mRNA is translated
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Figure 7-108 Molecular Biology of the Cell (© Garland Science 2008)
Interaction of Cap and polyA
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‫‪THE IRES‬‬
‫‪IRES = internal ribosome binding site‬‬
‫‪ ‬בעזרת ‪ IRES‬ניתן לדלג על ה‪ MET-‬הראשון ולהתחיל את התרגום‬
‫בנקודות אחרות‪ .‬זהו רצף של מס' מאות בסיסיםשיוצרים בד"כ מבנה‬
‫אופייני‪.‬‬
‫‪ ‬רצף זה קושר אליו הרבה מפקטורי האיניציאציה (אך לא את כולם)‬
‫ובכך "עוקפים" את הצורך במבנה ה‪.CAP-‬‬
‫‪ ‬יש וירוסים משתמשים ב‪ IRES-‬כטקטיקה לתרגום ה‪ RNA-‬שלהם‪.‬‬
‫‪ ‬חלקם אפילו מייצרים פרוטאזה החותכת את פקטור ה‪ eIF4G -‬ובכך‬
‫חוסמים תרגום דרך ה‪ .CAP-‬כי החלק הנותר אינו ספציפי ל‪.CAP-‬‬
‫‪‬‬
‫‪30‬‬
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HOWEVER…Many RNA Viruses
have capped genomic RNAs similar
to eukaryotic host mRNAs
• Most eukaryotic mRNAs are capped at
the 5’ end during nuclear processing.
• The terminal 5’ phosphate is first
removed by a 5’ triphosphatase.
• Guanyltransferase transfers GMP from
GTP to the 5’ end of the mRNA to add
the GpppN cap structure.
• The 5’ terminal inverted G residue is
then modified by methylation.
• Many RNA viruses replicate in the
cytoplasm and must use a viral
dependent capping mechanism
supplied by the RNA-Dependent-RNA
Polymerase.
• The Cap structure, m7GpppN, is most
common in viral and mammalian
mRNAs.
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Figure 6-72 Molecular Biology of the Cell (© Garland Science 2008)
Three distinct stages of translation
Initiation
•Rate limiting step
•Requires hydrolysis of ATP and GTP
•Results in formation of a complex containing the mRNA, the
ribosome and the initiator Met-tRNA
A. 5’ end (Cap) dependent initiation
• The initiation complex binds to the 5’ cap structure and
scans in a 5’ to 3’ direction until initiating AUG is
encountered
B. Internal ribosome entry
• Initiation complex binds upstream of initiation codon
C. Internal ribosome entry
•IRES
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Initiation factors in Eukaryotes
eIF-1
Component of the pre-initiation complex
eIF-1A
Component of the pre-initiation complex
eIF-2
Binds to the initiator tRNAMet within the ternary complex component of the preinitiation complex; phosphorylation of eIF-2 results in a global repression of
translation
eIF-3
Component of the pre-initiation complex; makes direct contact with eIF-4G and so
forms the link with the cap binding complex
eIF-4A
Component of the cap binding complex; a helicase that aids scanning by breaking
intramolecular base pairs in the mRNA
eIF-4B
Aids scanning, possibly by acting as a helicase that breaks intramolecular base pairs in
the mRNA
eIF-4E
Component of the cap binding complex, possibly the component that makes direct contact
with the cap structure at the 5 end of the mRNA
eIF-4G
Component of the cap binding complex; forms a bridge between the cap binding
complex and eIF-3 in the pre-initiation complex; in at least some organisms, eIF-4G
also forms an association with the poly(A) tail, via the polyadenylate-binding protein
eIF-5
Aids release of the other initiation factors at the completion of initiation
eIF-6
Associated with the large subunit of the ribosome; prevents large subunits from
attaching to small subunits in the cytoplasm
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‫‪STABILITY‬‬
‫‪MRNA‬‬
‫‪ ‬באופן כללי‪ mRNA ,‬אאוקריוטי יציב יותר מאשר פרוקריוטי‪.‬‬
‫‪ ‬קיימת שונות גדולה בזמן מחה"ח של ‪.mRNA‬‬
‫‪ ‬ככל שה‪ poly-A-‬ארוך יותר כך יציבות ה‪ mRNA-‬גבוהה יותר‬
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Figure 7-109 Molecular Biology of the Cell (© Garland Science 2008)
ELONGATION
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Elongation
eEF1a
GTP
Ribosome selects
aminoacylated tRNA
eEF1a and GTP are bound to
aminoacylated tRNA
P A
Ribosome catalyzes formation
of a peptide bond
Translocation is dependent on
eEF2 and GTP hydrolysis
Many ribosomes may translate
mRNAs simultaneously on the
same strand.
eEF2
GTP
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TERMINATION
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Termination
•Translation is terminated
at one of three stop codons
(UAA, UAG & UGA).
• Termination codon at the
A site is recognized by the
release factor instead of a
tRNA
• The release factor binds
the termination codon
• The peptide chain is then
released followed by
dissociation of the tRNA
and the ribosome
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DIFFERENCE IN INITIATION BETWEEN
PROKARYOTIC AND EUKARYOTIC
TRANSLATION
 Many
more factors in eukaryotes.
 Small
subunit binding site: Shine-Delgarno (pro)
vs. 5’ end (euk).
 Scanning
of the 5’utr in eukaryotes vs. direct
interaction with the AUG in prokaryotes.
 First
amino acid+tRNA: formyl Met with initiator
tRNA vs. Met and tRNA met.
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‫אנטיביוטיקה‬
‫‪44‬‬
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Evidence for the A and P sites: puromycin
•puromycin resembles an aminoacyl-tRNA
•it was used to show that fMet-tRNAfMet goes to the P site
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The puromycin reaction as an assay for peptidyl transferase
peptidyl puromycin is release from the
ribosome after peptide bond formation
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‫קיקיון מצוי‬
‫** מהזרעים מפיקים שמן קיק שהוא חומר רפואי משלשל‪.‬‬
‫משמש גם כשמן סיכה למכונות‪ .‬כיום נבדקת האפשרות‬
‫לנצל שמן זה לייצור תחליף לדלק למנועי דיזל‪.‬‬
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‫"וימן אלוהים תולעת‪ ...‬ותך את הקיקיון וייבש"‬
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Figure 6-77 Molecular Biology of the Cell (© Garland Science 2008)
Translational frameshift:
Allows more then one protein to be synthesized from a single mRNA. Retroviruses,
members of a large group of eukaryotic infecting pathogens, commonly use
translational frameshift in order to make the caspid, the RT enzyme and integrase
from the same transcript.
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Figure 6-78 Molecular Biology of the Cell (© Garland Science 2008)
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