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 polynucleotide 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

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


Exosome core proteins 6 x rnase ph 3 x s1 kh

Exosome Core Proteins: 6 x RNase PH + 3 x S1/KH

E. ColiArcheal Yeast Human

PNPaseRrp41Rrp41p/Ski6phRrp41pRNase PH Q17533

(x 3)(x 3)Rrp46phRrp46pRNase PH (Crn-5)

Mtr3phMtr3pRNase PH

Rrp42Rrp42phRrp42pRNase PH NP_508024

(x 3)Rrp43pOIP2RNase PH

Rrp45pPM/Scl-75RNase PH T28842

Csl4Csl4p/Ski4phCsl4pS1 RBD

Rrp4pRrp4phRrp4pS1/KH

Rrp40phRrp40pS1/KH

Other common proteins:

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

(RNase D?)Rrp6pPM/Scl-100RNase 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.


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