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[anubis2005]

A gateway cloning expression vector question


How do you know your gene will be transcribed and translated using the correct strand of DNA:

For instance:  You don't want this

TATATAA..ATGCTCCCG
...............TACGAGGGC


you want this

TATATAA..TACGAGGGC
...............ATGCTCCCG

Right?  You want the template strand to be on the same strand of the plasmid where the RNA polymerase binds to.

              
How do you know which strand the RNA polymerase will bind to, and how do you ensure your template is on that strand

Edited by anubis2005, 24 July 2009 - 01:27 PM.

[phage434]

Nope, this is wrong.  Although the 'template' strand is the bottom strand, the sequence created in the mRNA, which is the sequence used to make protein, is a sequence corresponding to the top strand, and it must contain the ATG start codon.  The bottom strand, which reads 3' to 5', would not even have the ATG in the correct 5' to 3' direction.  The RNA polymerase binds to the 'template' strand, moving in the 3' to 5' direction along that strand, and (like all biological enzymes), copies, creating an mRNA in the 5' to 3' direction.  The sequence of this copy will be the same (modulo T->U conversion) as the sequence of the top strand in your example.