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Protein Synthesis 2 Major topics covered: Translation: initiation, elongation and termination Comparison of eukaryotic translation to prokaryotic Medical relevance of translation: two points . c ontact info: David A. Schneider, Ph.D. Department of Biochemistry and Molecular Genetics - PowerPoint PPT Presentation
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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 [email protected] #: 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) Initiation2) Elongation3) Termination
A general cartoon of the
translation process
In bacteria, the first codon in the mRNA (AUG) leads to initiation and recruitment of the formyl-methionyl tRNA
So, where does this start?
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).
How does the ribosome find the first codon?
The “Shine-Delgarno” sequence in the mRNA:
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 UAGRF-2 = recognizes UAA and UGARF-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
Consequence: translation mechanisms are different, primarily at the initiation step
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
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) tRNAi
Met-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?