5 replies to this topic

[DzDz]

Question to the one that is currently up to date with knowledge about transcription   -

  I am going to clone and analyse a 5 ` regulatory region of a human gene. My questions are?

Where exactly the TSS lies – my prediction is that in the gene I analyse it is a little upstream ~40bp of the beginning of the known UTR from ENSEMBL and NCBI – does TSS always lie at beginning of the UTR? Can I trust the data from ENSEMBL – could the UTR mentioned there be longer? I found a common core promoter element Inr 40bp upstream of known UTR and it is known that  it should encompass the TSS. There is also a DPE  motif 30bp downstream so as it is supposed to be. I don`t know if my predictions are in opposite of the ENSEMBLE data. There are 3 options:

1. The end of the known 5` UTR is the TSS and I am wrong
2. The TSS lies upstream of the UTR and it is normal
3. The UTR is longer than the one in database

What you think could be true and how can I check it myself?? Please help

[dpo]

DzDz, on Jan 30 2009, 01:26 PM, said:

Question to the one that is currently up to date with knowledge about transcription   -

  I am going to clone and analyse a 5 ` regulatory region of a human gene. My questions are?

Where exactly the TSS lies – my prediction is that in the gene I analyse it is a little upstream ~40bp of the beginning of the known UTR from ENSEMBL and NCBI – does TSS always lie at beginning of the UTR? Can I trust the data from ENSEMBL – could the UTR mentioned there be longer? I found a common core promoter element Inr 40bp upstream of known UTR and it is known that  it should encompass the TSS. There is also a DPE  motif 30bp downstream so as it is supposed to be. I don`t know if my predictions are in opposite of the ENSEMBLE data. There are 3 options:

1. The end of the known 5` UTR is the TSS and I am wrong
2. The TSS lies upstream of the UTR and it is normal
3. The UTR is longer than the one in database

What you think could be true and how can I check it myself?? Please help

4. You're both right. TSS are not fixed for all genes, several genes have relaxed TSS spread over a region of ~100 bp. If you want to be sure what the start sites are in the cell line/tissue you're interested in, you'll have to do 5'RACE and analyse several clones.

[DzDz]

Quote

4. You're both right. TSS are not fixed for all genes, several genes have relaxed TSS spread over a region of ~100 bp. If you want to be sure what the start sites are in the cell line/tissue you're interested in, you'll have to do 5'RACE and analyse several clones.

Thansk for reply -  but do TSS always mean a place where UTR starts or could it be some bp before if yes how many bp before ? Other words - UTR is also "relaxed" or only TSS I ask cuase I believe in UTR determination from capped mRNA in ENSEMBL and also believe in my predictions:)

[dpo]

DzDz, on Jan 30 2009, 01:56 PM, said:

Thansk for reply -  but do TSS always mean a place where UTR starts or could it be some bp before if yes how many bp before ? Other words - UTR is also "relaxed" or only TSS I ask cuase I believe in UTR determination from capped mRNA in ENSEMBL and also believe in my predictions:)

The TSS is the first nucleotide of the UTR (at least I think so, I don't think there's any gene which immediately begins with ATG), so yes, UTRs can also be 'relaxed' and differ in length.

[DzDz]

dpo, on Jan 30 2009, 01:58 PM, said:

The TSS is the first nucleotide of the UTR (at least I think so, I don't think there's any gene which immediately begins with ATG), so yes, UTRs can also be 'relaxed' and differ in length.

A little misunderstanding -I know that UTR must be there but the question is if  the TSS where the pre-initiation complex binds could be some bp before the UTR or it binds and imeditialy produces UTR sequence? Inr encompasess TSS so could it be 40bp upstream of the sequence coding the begining of the 5` UTR?

[dpo]

DzDz, on Jan 30 2009, 02:33 PM, said:

dpo, on Jan 30 2009, 01:58 PM, said:

The TSS is the first nucleotide of the UTR (at least I think so, I don't think there's any gene which immediately begins with ATG), so yes, UTRs can also be 'relaxed' and differ in length.

A little misunderstanding -I know that UTR must be there but the question is if  the TSS where the pre-initiation complex binds could be some bp before the UTR or it binds and imeditialy produces UTR sequence? Inr encompasess TSS so could it be 40bp upstream of the sequence coding the begining of the 5` UTR?

To me, the definition of a TSS is the first nucleotide of the 3'UTR. I'm not really familiar with the positions where the pre-initiation complex binds, so I'll have to skip that question I'm afraid. Maybe somebody else is more acquainted with this?