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Thread: uncut plasmid

  1. 27-10-2009, 11:22 PM #1
    motivationG
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    uncut plasmid

    why do uncut plasmids cause RNA polymerase to stop?
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  2. 27-10-2009, 11:53 PM #2
    zhangyuzhi
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    Quote Originally Posted by motivationG View Post
    why do uncut plasmids cause RNA polymerase to stop?
    because of supercoiling?
    2005: MBChB Medical Studies, Chinese University of Hong Kong (Quitted)
    2005-2010: MSci Biochemistry with a Year in Industry/Research, Imperial College London
    2010- ? : MBBS Medicine, Imperial College London
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  3. 27-10-2009, 11:58 PM #3
    motivationG
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    Quote Originally Posted by zhangyuzhi View Post
    because of supercoiling?
    what is supercoiling? and why does this stop transcription?
    Thanks so much for your quick response!!
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  4. 28-10-2009, 12:02 AM #4
    zhangyuzhi
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    Quote Originally Posted by motivationG View Post
    what is supercoiling? and why does this stop transcription?
    Thanks so much for your quick response!!
    supercoiling is the formation of compact DNA structures. grab a genetics/molecular biology text and they have detailed descriptions there. including diagrams (very important lol).

    to be honest I am not sure whether this will suppress transcription coz to my knowledge, the RNA polymerase I know (T7 RNA pol) does work on uncut plasmids...

    my guess would be the compactness of DNA restricts accessibility of the enzyme to the sequence, therefore it cannot transcribe.
    2005: MBChB Medical Studies, Chinese University of Hong Kong (Quitted)
    2005-2010: MSci Biochemistry with a Year in Industry/Research, Imperial College London
    2010- ? : MBBS Medicine, Imperial College London
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  5. 28-10-2009, 12:04 AM #5
    motivationG
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    Thanks so much!!
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  6. 28-10-2009, 12:20 AM #6
    motivationG
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    Quote Originally Posted by zhangyuzhi View Post
    supercoiling is the formation of compact DNA structures. grab a genetics/molecular biology text and they have detailed descriptions there. including diagrams (very important lol).

    to be honest I am not sure whether this will suppress transcription coz to my knowledge, the RNA polymerase I know (T7 RNA pol) does work on uncut plasmids...

    my guess would be the compactness of DNA restricts accessibility of the enzyme to the sequence, therefore it cannot transcribe.

    can i also ask why uncut plasmid can be nicked when it is actually "uncut"?? What cut it to make it nicked?
    Hope u understand my question
    Thanks
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  7. 28-10-2009, 12:32 AM #7
    zhangyuzhi
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    Quote Originally Posted by motivationG View Post
    can i also ask why uncut plasmid can be nicked when it is actually "uncut"?? What cut it to make it nicked?
    Hope u understand my question
    Thanks
    the nick is with respect to one strand only right?

    enzymes such as topoisomerase I will nick one strand of the plasmid to relax tension, which will cause the formation of nicked circular plasmid. sometimes mechanical shearing does the same.

    uncut means it is still circular. only a double-strand break will turn it into a linearized form.
    2005: MBChB Medical Studies, Chinese University of Hong Kong (Quitted)
    2005-2010: MSci Biochemistry with a Year in Industry/Research, Imperial College London
    2010- ? : MBBS Medicine, Imperial College London
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