2 replies to this topic

[albatross]

When a DNA fragment (a compelte ORF) is cloned to a expression vector by double blunt end ligation, and if the coding strand is on the other strand relative to the promoter,
-------------promoter---blunt-------------------------------------blunt--------
----------------------- ---blunt--ATG>>>>>>>>Stop codon---blunt--------
how could the RNA pol choose the correct template strand for transcription?

Edited by albatross, 26 August 2009 - 09:06 PM.

[Penguin]

albatross, on Aug 27 2009, 06:04 AM, said:

When a DNA fragment (a compelte ORF) is cloned to a expression vector by double blunt end ligation, and if the coding strand is on the other strand relative to the promoter,
-------------promoter---blunt-------------------------------------blunt--------
----------------------- ---blunt--ATG>>>>>>>>Stop codon---blunt--------
how could the RNA pol choose the correct template strand for transcription?

Hi,
The coding strand has the same sequence as the mRNA except the Ts are now Us. The template strand is complementary to the coding strand and so when RNA pol binds to its promoter on the template strand it will produce complementary mRNA which corresponds to the coding strand,

Hope this is clear,
P

[swanny]

albatross, on Aug 27 2009, 03:04 PM, said:

When a DNA fragment (a compelte ORF) is cloned to a expression vector by double blunt end ligation, and if the coding strand is on the other strand relative to the promoter,
-------------promoter---blunt-------------------------------------blunt--------
----------------------- ---blunt--ATG>>>>>>>>Stop codon---blunt--------
how could the RNA pol choose the correct template strand for transcription?

Think about the chemistry of DNA. If the gene was cloned on the opposite strand to the promoter, it would be in the reverse direction to the prooomoter; the  RNA pol would simply transcribe the same strand as the promoter. When the transcript hit the ribosome, something would probably be translated, up the appearance of a stop codon, but it's doubtful it would make anything meaningful.

-------------promoter---3'                                                5'--------           Vector
----------------------- ---5'                                                3'--------

                                 5'-------------------------------------3'                        Insert
                                 3'-Stop codon<<<<<<<<GTA----5'


-------------promoter---blunt-------------------------------------blunt--------
----------------------- ---blunt-Stop codon<<<<<<<<GTA----blunt--------