Design the elegant experiment
Analyze the experimental results
Answer the scientific question
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Establish a good model
Study the peptidyl transferase reaction
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Antibiotics inhibit cell division by blocking specific steps in translation—a powerful tool to fight disease
Targets of many antibiotics are translational machinery
First question:
Which part of ribosome is responsible for peptidyl transferase reaction? (50S subunit or 30S subunit?)
Localization of peptidyl transferase
Example I:
Use puromycin to study the peptidyl transferase reaction and prove that ribosome is a ribozyme(核酶)
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Basic conception: what is peptidyl transferase reaction?
Remove 30S subuint
[14C]poly(Phe)
50S subunit/poly(Phe)-tRNA Detection of the 14C-labeled released peptidylpuromycin can reflect the peptidyl transferase activity
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Overview of the events of translation
Initiation
Termination
Elongation
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链霉素 四环素
氯霉素 红霉素
氯霉素
Molecular target: peptidyl transferase reaction center of 50S subunit
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N-terminal
New peptide bond
C-terminal
Peptidyl transferase reaction (肽酰转移酶反应)
Raise scientific question about peptidyl transferase reaction (提出科学问题）
Which part of ribosome is responsible for peptidyl transferase reaction? (50S subunit or 30S subunit?)
We should establish a system which can detect peptidyl transferase
activity easily and in vitro(体外)
Puromycin can be used to detect the peptidyl transferase reaction in vitro
Puromycin (嘌呤霉素) causes polypeptide synthesis to terminate prematurely
Release of peptidyl-puromycin from ribosome
Use puromycin to explore the translational mechanism:
Next question: rRNA or protein is responsible for
peptidyl transferase reaction?
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How to answer the scientific question(解决科学问题)
Establish a good model
Inhibit peptidyl transferase reaction specifically
四环素
Molecular target: A site of 30S subunit
Inhibit aminoacylated-tRNA binding to A site
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In addition to medical benefits, antibiotics can play important role in research for understanding the function of translational machinery
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Puromycin (嘌呤霉素)
Puromycin resembles the aminoacylated-tRNA, so it can bind to the A site of ribosome
Puromycin (嘌呤霉素)
Puromycin
肽酰嘌呤霉素 (peptidylpuromycin)
Model: poly(U)-ribosome-poly(Phe) (14C-labeled) UUU--Phe poly(U)—poly(Phe)
放射性标记(14C-labeled)
Release of peptidyl-puromycin from ribosome
Detection of the 14C-labeled released peptidyl-puromycin can re activity
50S subunit of ribosome alone can have peptidyl transferase activity
[14C]