Transcript Initiation in eukaryotes

1
Generalized G protein cycle
(GEF)
2
Make a list of translation factors that are G-proteins, or GEFs
initiation
3
elongation
termination
4
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
5
6
7
‫‪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.
9
10
11
12
Figure 6-39a Molecular Biology of the Cell (© Garland Science 2008)
13
INITIATION IN
EUKARYOTES
14
‫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.‬‬
‫‪17‬‬
‫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
20
21
Figure 7-107 Molecular Biology of the Cell (© Garland Science 2008)
‫ופקטורים נוספים‬
‫‪22‬‬
23
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
24
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
25
26
Figure 7-108 Molecular Biology of the Cell (© Garland Science 2008)
Interaction of Cap and polyA
27
28
29
‫‪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
31
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.
32
33
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
34
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
35
‫‪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
‫‪36‬‬
37
Figure 7-109 Molecular Biology of the Cell (© Garland Science 2008)
ELONGATION
38
39
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
40
TERMINATION
41
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
42
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.
43
‫אנטיביוטיקה‬
‫‪44‬‬
45
46
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
47
The puromycin reaction as an assay for peptidyl transferase
peptidyl puromycin is release from the
ribosome after peptide bond formation
48
49
50
51
‫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.‬‬
‫‪52‬‬
‫"וימן אלוהים תולעת‪ ...‬ותך את הקיקיון וייבש"‬
53
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.
54
Figure 6-78 Molecular Biology of the Cell (© Garland Science 2008)
55