Term issue: Spt5 CTD-T1(UnP)

[ValWood]

O13936 Spt5 CTD-T1(UnP) (in the consensus TPAWNSGSK C-terminal domain repeat) For the recruitment of ~Csk1~ Ceg1 by Spt5

PMID:24939935 How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes. "Thus, Thr1 phosphorylation elicited an 18-fold decrement in affinity of the fission yeast GTase for the Spt5 CTD."

[nataled]

Hi Val, Two questions: 1) I suspect you need T1(unP), not W4...? 2) There exists in the sequence actually only one instance of that "consensus" but multiple instances of shorter sequences:

TPAWNSGSK  1
TPAWNSGS   2
TPAWNSG    5
TPAWNS     5
TPAWN     10

The reason I ask is because I need to know if I need to make a term specific to the one instance of that sequence, or if I have to allow for multiple possibilities with unknown status. This would be similar to how PR:000035411 is defined. In this case, do all the T1 positions need to be unphosphorylated for functionality, or is one enough...?

[ValWood]

1. Sorry yes T1.

2. I thought the authors had chosen a strange 'consensus" but I think if the repeats are aligned these are the most common residues at each position (even though they are only all together in one repeat)

3. Tricky, To evaluate the impact of Spt5 CTD phosphorylation, we tested the ability of the unphosphorylated and Thr1- phosphorylated 22-amino-acid synthetic peptides SGSK TPAWNSGSKTPAWNSGSK and SGSK(Tp)PAWNSGSK (Tp)PAWNSGSK to compete with a fluorescein-tagged 22-mer Spt5 CTD peptide for binding to Pce1 in a fluorescence polarization assay. An initial Pce1 titration showed that the Kd for the 22-mer fluorescent Spt5 CTD containing two complete nonamer repeats was 109 6 5 mM (Fig. 5B); this value was 90-fold higher than the Kd of Pce1 for the much longer Spt5-(801–898) ligand containing 10 nonamer repeats (Fig. 4C). This acute dependence on the number of nonamer repeats for high-affinity binding of Pce1 to Spt5 CTD in the fluorescence assay is in accord with results obtained using a His tag affinity chromatography assay for protein–protein interaction, which showed that Pce1 bound well to an Spt5 CTD composed of 10 nonamer repeats, less well to a six-nonamer array, and not detectably to a Spt5 CTD with only four nonamer elements (Pei and Shuman 2002).

So it seems the more the merrier....I guess multiple possibilities with unknown status?

[nataled]

Hmm, makes me wonder if something like "unphosphorylated Thr on at least six repeats" would need to be stated? Difficult indeed, because natural sets of repeats in vivo could behave a bit differently. I also note that the Pei and Shuman 2002 paper states "As few as four nonamer repeats suffice for Spt5 binding to Pct1 in vitro, whereas six repeats are required for Spt5 binding to Pce1." Pce1 is the same protein studied in your cited paper.

By the way, a later paper from the same group (PMID:25414009) indicates the important part of the consensus is TPAWN.

I'll work something up and have you take a look.

[ValWood]

Pei and Shuman is next on my list!

[nataled]

How is this? The definition is drawn from the three papers mentioned (your original, the newer paper from the same authors, and Pei).

[Term]
id: PR:000085736
name: transcription elongation factor spt5 unphosphorylated 1 (Schizosaccharomyces pombe)
def: "A transcription elongation factor spt5 (Schizosaccharomyces pombe) that lacks phosphorylation on the Thr residue of at least six C-terminal domain TPAWN consensus sequences." [PRO:DAN, PomBase:VW, PMID:24939935, PMID:11893740, PMID: 25414009]
comment: Category=organism-modification. Requested by=PomBase. Note: The spt5 CTD is typically defined as a nonapeptide, TPAWNSGSK. However, that precise peptide occurs only once in the spt5 sequence, and only the TPAWN section is important for interaction [PMID:25414009]. The need for six copies of the consensus is inferred from PMID:11893740.
synonym: "Spom-spt5/UnPhos:1" EXACT PRO-short-label [PRO:DNx]
is_a: PR:000085735 ! transcription elongation factor spt5 (Schizosaccharomyces pombe)
relationship: only_in_taxon NCBITaxon:4896 ! Schizosaccharomyces pombe

[ValWood]

Sounds great!