translation iniation - cloning (Nov/24/2004 )

Hi,
There is an in-frame start codon upstream of the "official" start codon in my target gene. I have cloned a fragment that includes both - will translation be initiated at the first start codon after the promoter, or is there some form of cryptic signal to initiate translation only at "correct" start codons?

Hi.
I think that there is a certain initiation signal upstream of the start codon such that the ribosome will bind there.
Once ribosome bind to the initiation signal, the ribosome will scan through the start codon and start the translation.
Therefore, I guess that it depends on where your initiation signal located.
Dunno this helps or not.

regards.

Hi,
I think the expression vector will contain a ribosome binding site - I'll have to check that out.
Thanks

its called a shine delgarno site / sequence. Presumably you will need to know which is the true start (both may even be used?!) at some point. The best way to do this is to look at the RNA. If its a prokaryote you could use primer extension or S1 mapping. Otherwise you could use RT-PCR to work it out. to do this you would design one primer that anneals to the RNA pointing back up towards the transcriptional start site (primerA). You would then need a reverse primer between start site 1 and primerA and another between the two start codons?