Domain Analysis of the Chloroplast Polynucleotide Phosphorylase Reveals Discrete Functions in RNA De...
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Domain Analysis of the Chloroplast Polynucleotide Phosphorylase Reveals Discrete Functions in RNA Degradation, Polyadenylation, and Sequence Homology with Exosome Proteins. The Plant Cell , Vol. 15, 2003-2019, September 2003

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The plant cell vol 15 2003 2019 september 2003

Domain Analysis of the Chloroplast Polynucleotide Phosphorylase Reveals Discrete Functions in RNA Degradation, Polyadenylation, and Sequence Homology with Exosome Proteins

The Plant Cell, Vol. 15, 2003-2019, September 2003

Shlomit Yehudai-Resheffa, Victoria Portnoya, Sivan Yogeva, Noam Adirb and Gadi Schuster1

Mike 7/14


The function of spinach chloroplast p olynucleotide p hosphorylase pnpase
The function of spinach chloroplast Phosphorylase Reveals Discrete Functions in RNA Degradation, Polyadenylation, and Sequence Homology with Exosome Proteinspolynucleotide phosphorylase (PNPase)

1.chloroplast mRNA degradation (chloroplast and bacteria)

1). endonucleolytic cleavage

2). addition of poly(A)-rich sequences to the endonucleolytic cleavage products

(PAP in E.coli)

3). exonucleolytic degradation

2.PNPase in the chloroplast was found to form a homotrimeric complex

and lacks any known interactions with other proteins.

RNA. 7, (2001), 1464–1475

3.No PAP can be detected in spinach chloroplasts, and thus both

polyadenylation and degradation are performed by one enzyme,

PNPase

Mol. Cell. Biol.21, (2001), 5408–5416


The spinach chloroplast pnpase structure is similar to that of the bacterial enzyme

core = RNase PH domain Phosphorylase Reveals Discrete Functions in RNA Degradation, Polyadenylation, and Sequence Homology with Exosome Proteins

The spinach chloroplast PNPase structure is similar to that of the bacterial enzyme

The amino acid sequence and domain structure is largely conserved between bacteria and organelles.


Rna degradation and polyadenylation activities of the spinach chloroplast pnpase and its domains
RNA degradation and polyadenylation activities of the spinach chloroplast PNPase and its domains

# bacteria : domain 2 have activity only

Degradation : domain 1 and 2

Polymerization : domain 2

Product : NDP (TLC)


The high affinity poly a binding site is located in the s1 domain
The high-affinity poly(A) binding site is located in the S1 Domain

UV light cross-linking assay

(Lisitsky et al., 1997b; Lisitsky and Schuster, 1999).

UV light cross-linking competition assay

substrate : 32P-psbA RNA

domain 2 (only)


The plant cell vol 15 2003 2019 september 2003
Unlike the FL PNPase, the proteins that include only one core domain do not pause at a stem-loop structure

PNPase enzyme is its pausing at a stem-loop structure when processively degrading RNA.

EMBO J. (1996) 15, 1132–1141


A platform of 6 to 12 nucleotides 3 to the stem loop is required for rna polyadenylation by pnpase
A platform of 6 to 12 nucleotides 3' to the stem loop is required for RNA polyadenylation by PNPase

E. coli PAP I is inhibited by a stem-loop structure but that the addition of two nucleotides 3' to the stem loop is sufficient to promote efficient polyadenylation.

Nucleic Acids Res.(2000) 28, 1139–1144.


The plant cell vol 15 2003 2019 september 2003
The spinach chloroplast PNPase and its active fragments complement the growth of an E. coli PNPase- and RNase PH–less Strain at 18°C

E. coli strain SK 8992


Summary
Summary complement the growth of an


Exosome core proteins 6 x rnase ph 3 x s1 kh
Exosome Core Proteins complement the growth of an : 6 x RNase PH + 3 x S1/KH

E. ColiArcheal Yeast Human

PNPase Rrp41 Rrp41p/Ski6p hRrp41p RNase PH Q17533

(x 3) (x 3) Rrp46p hRrp46p RNase PH (Crn-5)

Mtr3p hMtr3p RNase PH

Rrp42 Rrp42phRrp42p RNase PH NP_508024

(x 3) Rrp43pOIP2 RNase PH

Rrp45p PM/Scl-75 RNase PH T28842

Csl4 Csl4p/Ski4p hCsl4p S1 RBD

Rrp4p Rrp4phRrp4p S1/KH

Rrp40p hRrp40p S1/KH

Other common proteins:

(RNase R?) Rrp44p/Dis3p (hDis3p) RNase R

(RNase D?) Rrp6p PM/Scl-100 RNase D (Crn-3)(nuclear only)

Chloroplast

PNPase (x3 ?)

domain 2

domain 1

red: in vitro 3’-5’exonuclease activity

PNPase lacks any known interactions with other proteins.