Appropriate distance b2t rib binding site & ATG?

Stefan Gruenert (WCI) sg124 at mole.bio.cam.ac.uk
Wed Jun 1 12:56:56 EST 1994


Huanquan Zheng from Helsinki wrote:
>I wondered whether the distance of 15bp is suitable or not.
>
>any comment?
               
I assume we're talking about eukaryotic ribosomes here and the rib 
binding site is the cap structure on an eukaryotic message, yes ?
In that case there is a reference from big translation Mama Kozak in 
'Gene Expression (1991) Vol. 1, No. 2:pp 111-115, "A short leader 
sequence impairs the fidelity of initiation by eukaryotic ribosomes".
No wonder you never heard of the Journal as it is at least a bit obscure.
Therefore I describe briefly the findings: 
Experiments in vitro (wheat germ), 
capped message with two AUGs in the same reading frame, so you can 
measure how many ribosomes start at the first and second AUG.
The leader was 3, 6, 9, 12, 32 nt long before the first AUG.
She found that up to 12 nt initiation upstream was inefficient 
(about similar product yield from both AUGs), and with 32 nt she got 100% 
from upstream. 
My gut feeling from four years in vitro translation is she's right, even 
though she hid the paper in some inaccessible place for nobody to check 
the gels. So you might be a bit on the short side with your 15 nt long 
leader. But it depends what you want to do, I guess. For in vivo I'm sure 
you'll be fine with an even shorter leader (The shortest 5'untranslated 
region in a natural mRNA (known to me) is 7 nt), if you need only "some" 
expression.
But if it's not necessary why  risk it and put in an extra 15 nt.
Sorry for rambling on but it would be much easier if the question would 
be a bit more precise. For example if you're talking about internal 
ribosome entry like on EMCV, or polio mRNA, which carry a ribosome 
binding site forget what I said above and mail me for advise on these,
good luck to everybody reading this,
Stefan


Stefan Grunert         sg124 at mole.bio.cam.ac.uk
Wellcome/CRC
Cambridge/UK





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