Transcript Protein Synthesis 2

Protein Synthesis 2
Major topics covered:
•Translation: initiation, elongation and termination
•Comparison of eukaryotic translation to prokaryotic
•Medical relevance of translation: two points
related text:
Biochemistry
Garret and Grisham, 4th ed.
Chapter 30
contact info:
David A. Schneider, Ph.D.
Department of Biochemistry and Molecular Genetics
[email protected]
office #: 934-4781
Reminder of Friday’s lecture:
• Translation is the process of making protein
from an RNA template
• Fidelity is affected by several steps
• tRNAs are the “adapters” that translate the
4-nucleotide language of DNA/RNA into the
20-amino acid language of proteins.
• Aminoacyl-tRNA synthetases are ancient,
but accurate enzymes.
• Ribosomes are large, complicated
“machines”.
I will start with a general overview of translation
(the example is eukaryotic)
The ribosome is the ribozyme that catalyzes
peptide bond formation.
What other factors participate in translation,
and how is the whole process orchestrated?
The ribosome is the ribozyme that catalyzes
peptide bond formation.
What other factors participate in translation,
and how is the whole process orchestrated?
Translation consists of three steps:
1) Initiation
2) Elongation
3) Termination
A general
cartoon of the
translation
process
So, where does this start?
In bacteria, the first codon in the mRNA (AUG) leads to
initiation and recruitment of the formyl-methionyl tRNA
How does the ribosome find the first
codon?
The “Shine-Delgarno” sequence in the mRNA:
Base pairing between the Shine Dalgarno sequence and the 3´ end of 16S
rRNA facilitates translation initiation. Consequently, the efficiency of
translation initiation is determined by:
1) How well the S.D. sequence conforms to the consensus sequence that is
complementary to the 3´ end of 16S rRNA.
2) The distance between the S.D. sequence and the start codon (a 7 base
spacer is optimal).
Three translation initiation factors are required
(in addition to the ribosome and aa-tRNA)
The process of translation initiation in
prokaryotic cells
High translation initiation rates lead to multiple
ribosomes per message (“polysomes”)
Electron micrograph of polysomal mRNA
Note of interest: ribosome occupancy on mRNA plays a major role in
determining mRNA decay rate
The translation
elongation cycle
The chemistry of peptide bond formation
Translation terminates
when a stop codon
(UAA, UAG, UGA) enters
the A-site
Translation termination factors:
RF-1 = recognizes UAA and UAG
RF-2 = recognizes UAA and UGA
RF-3 = G-protein; helps trigger hydrolysis
(by the 23S rRNA)
RRF = liberates ribosome/release factors
Important term =
molecular mimicry
Translation factors use
molecular mimicry to
utilize common binding
sites on the ribosome
From Ramakrishnan, Cell 108: 557 (2002)
Translation is a cycle
(final overview)
A more detailed animation of translation, including factors
Translation is a highly conserved process
among all living things…
However, important differences exist
between bacteria and eukaryotes
(e.g. you!)
Important difference #1:
Ribosomes are substantially different
Important difference #2: mRNA is very different
in prokaryotes versus eukaryotes
Bacterial mRNA: lacks 5’ cap, poly-A tail not required, multiple orfs per transcript, SD sequence
eukaryotic mRNA: 7-MeG cap, poly-A tail, one orf per transcript, no sequence specific binding
Consequence: translation mechanisms are different, primarily at
the initiation step
The structural arrangement and
required factors for translation
initiation are substantially
different in eukaryotes,
compared to bacteria
Overview of
eukaryotic
translation initiation
Step 1: eIF1, 1A, 3 and 5 bind to 40S (not shown)
tRNAiMet-eIF2:GTP is recruited
Step 2: eIF4 proteins associate with mRNA
(cap and tail) and bind 43S
preinitiation complex
Scanning
Step 3: eIF5-mediated ejection of IFs and 60S binds
Translation elongation is very similar in eukaryotes
and prokaryotes
eEF1a
eEF1b
eEF2
Translation termination in
eukaryotes is
mechanistically similar to
prokaryotes…
Important difference:
only one release factor is
required
What have we learned (lectures 1&2)?
•tRNAs “adapt” the 4-base nucleotide code to a 20 amino
acid protein code.
•Charging of tRNAs and codon:anticodon interactions are
critical for fidelity.
•Ribsomes are big-big-big ribozymes… that we can now
visualize in some detail.
•Translation is a complicated process that is geared to be
efficient and accurate!
•Eukaryotic translation varies from prokaryotic translation
most significantly at the initiation step.
We know that translation and ribosome composition varies
between bacteria and eukaryotic cells
Why does this matter?
We know that translation and ribosome composition varies
between bacteria and eukaryotic cells
Why does this matter?
Fungi and bacteria often occupy the same environment and
battle for the same resources. Thus, they try to kill each
other
We benefit!
Several common
antibiotics with
mode of action and
molecular target
(of some) mapped
Note: your mitochondrial ribosomes are similar to those of bacteria,
thus some toxicity occurs
Puromycin is a charged tRNA (tRNATyr) analog:
as expected it inhibits translation in all organisms
Many human genetic disorders originate from
nonsense mutations
Nonsense mutations: premature stop codons in orf leading to
termination of translation and incompletely synthesized protein
Many human genetic disorders originate from
nonsense mutations
Nonsense mutations: premature stop codons in orf leading to
termination of translation and incompletely synthesized protein
PTC124 has progressed effectively through Phase 2
clinical trials and can rescue CFTR mRNA levels
-Kerem, et al. The Lancet (2008)
THE END
-any questions?