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Can anyone help with this RNA question

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Can anyone help with this RNA question

Postby georgina009 » Wed Mar 12, 2008 12:04 am

i was given a strand of E. coli mRna and I can't figure out where the promoter and pribnow box are.
It looks like this:

TACTCCACAT AATTGTCCTT CTTTTTAGAG GAAAAATG

So it ends in the start codon and the Shine dalgarno sequence is GAGGA (I think, correct me if I'm wrong) but where is the promoter and pribnow?
Thanks in advance
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Re: Can anyone help with this RNA question

Postby blcr11 » Wed Mar 12, 2008 3:35 pm

If this is mRNA, why are there Ts in the sequence? This is supposed to represent the promoter region maybe? The promoter isn’t transcribed into message. It lays upstream of the transcriptional initiation site, which is not the same thing as the translational start site. Bacterial promoter regions are generally composed of two very roughly conserved features. The Pribnow Box sits 5-8 bases upstream of the transcriptional start site (almost always an A or G), while the +35 region sits roughly 30-40 bases upstream of the transcriptional start site. The most highly conserved part of the +35 region is a TTG sequence, but there is a lot of room for different sequences. One of the best studied promoters, the lac promoter, doesn’t have a TTG sequence, for example. There may not be (in fact, probably won't be) an exact fit to the consensus. I don’t think the Shine-Dalgarno sequence should be in this stretch of nucleic acid, if this is supposed to be the promoter region. Shine-Dalgarno should be farther downstream, closer to the initiator methionine. It would be a very unusual gene to have the initiator ATG so close to its promoter.

My best guess for the position of the +35 and Pribnow Boxes would be:

TACTCCACAT AATTGTCCTT CTTTTTAGAG GAAAAATG

You can’t really be sure, though. You can make a case for other assignments, but this one is gives enough room for properly spacing both elements. This is placing the elements by rough sequence similarity and position relative to a presumed transcriptional start site. There are typically 11-15 bases between the 5’ end of the +35 region and the 3’ end of the Pribnow Box.

Edit: Sorry, I said that wrong. There are 11-15 bases between the 3' end of the +35 region and the 5' end of the Pribnow Box. Previously, I reversed the 3'/5' order by mistake.
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Postby georgina009 » Wed Mar 12, 2008 11:18 pm

thanks for the help. So is the shine dalgarno sequence likly to be what i said? (GAGGA)
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Re: Can anyone help with this RNA question

Postby blcr11 » Thu Mar 13, 2008 1:46 pm

That all depends. Is the 38 or so bases you've been given supposed to be a promoter region, or is it supposed to be part of the message? If it part of the message, and the ATG on the 3'-end is the initiator Met, then yes, it could be a Shine-Dalgarno sequence. But--and I say--but, if the 38 or so bases is supposed to represent a promoter region, then no, probably it is not a Shine-Dalgarno sequence.
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Re: Can anyone help with this RNA question

Postby georgina009 » Mon Mar 17, 2008 5:34 am

Sorry I only wrote a short bit. I found an area of TTGACA but it is nucleotides 86-91 and the start codon is 226. Then what i think is the pribnow box is CATAAT which is nucleotides 198-123. So i guess what I really what to know is: is the TTGACA got anything to do with it so that if it were removed would it impact on transcription and translation. Thanks
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Postby georgina009 » Mon Mar 17, 2008 5:35 am

edit - the pribnow box is nucleotides 198-203
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