RNGTT: Human RNA guanylyltransferase and 5'-phosphatase

Target ID:

RNGTTA

Aliases:

CAP1ADKFZp686J2031hCAPHCEHCE1

Deposition Date:

Saturday 15th October 2005

Families:

Phosphatases

Structure type:

apo

Download Coordinates:

2C46

Authors:

J.DEBRECZENI,C.JOHANSSON,E.LONGMAN,O.GILEADI,P.SAVITSKYSMEE,C.SMEE,G.BUNKOCZI,E.UGOCHUKWU,E.VON DELFT,M.SUNDSTROM,J.WEIGELT,C.ARROWSMITH,A.EDWARDS,S.KNAPP

Site:

Oxford

ICM Datapack:

ICM Datapack

2C46

tabs

Structure Details

RNA triphosphatase catalyzes the hydrolysis of the gamma-phosphate of nascent pre-mRNA to form a diphosphate end which is subsequently capped with GMP by RNA guanyltransferase and methylated by (guanine-7) methyltransferase to yield mature m-RNA. These three enzymatic activities are present as separate polypeptides in yeasts. Metazoans including humans contain 2 genes: a separate cap methyltransferase (RNA guanine-7-methyltransferase, RNMT), and a bifunctional capping enzyme (RNGTT) encoded by a single gene exhibiting both RNA 5â€™-triphosphatase and guanylyltransferase activities.

RNGTTA contains an HCxxxxxR(S/T) motif typical for the cysteine phosphatase superfamily. RNA phosphatases are divided into two subgroups: The triphosphatase domains of bifunctional plant and metazoan capping enzymes which exclusively hydrolyze g-phosphates and monofunctional enzymes that convert RNA triphosphate ends to 5â€™ diphosphates and 5â€™ monophosphates.

Primary structures of RNA-specific phosphatases of different organisms show high sequence similarity but differ from other cysteine phosphatases outside the HCxxxxxR(S/T) motif.

RNA capping is essential for cell growth, and mutations of the triphosphatase, guanylyltransferase, or methyltransferase components of the capping apparatus that abrogate catalytic activity are lethal in vivo. The m7G cap facilitates translation initiation and failure to cap pre-mRNAs results in their accelerated decay by exo-ribonucleases.

Recently it has been shown that cap formation is an essential component of a transcription elongation checkpoint critical to HIV gene expression. Functional cap formation is stimulated in infected cells by HIV-Tat, a small RNA-binding protein required for efficient transcription of HIV genes. It has been shown that TAT i nteracts with RNGTTA and stimulates its guanylyltransferase and triphosphatase activitity suggesting that this complex contributes to the activation of HIV gene expression.

RNGTT

PDB:2C46

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|4506563
Entry Clone Source:MGC
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:E. coli BL21(DE3).

Construct

Prelude:
Sequence:
Vector:pLIC-SGC1

Growth

Medium:
Antibiotics:
Procedure:Two litre of TB + 50 µg/mL Kanamycin in two 2.5-L baffled flasks were inoculated with 10 mL overnight culture. The culture was grown at 37°C and transfered to a 18°C shaker at an OD of 3.3. Protein expression was induced with 1 mM IPTG for 12 h. The cells were then collected by centrifugation and frozen at -80°C.

Purification

Procedure
Column 1 : Ni-affinity chromatography.
Buffers: Lysis buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP. Wash buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 50 mM imidazole, 0.5 mM TCEP. Elution buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 250 mM imidazole, 0.5 mM TCEP.
Procedure : The cell extract was loaded on the column at 0.8 mL/minute on an AKTA-express system (GE/Amersham). The column was washed with 10 volumes of lysis buffer and 10 volumes of wash buffer. The his-tagged protein was eluted with a liner gradient of elution buffer at 0.8 mL/min. The eluted peak recorded at 280nm was automatically collected.
Column 2: Size exclusion chromatography Hiload 16/60 Superdex 75 prep grade 120 mL, Code no. 17-1069-01 Amersham Biosciences.
Buffers : SEC- buffer : 10 mM HEPES, pH 7.5, 500 mM NaCl, 0.5 mM TCEP.
Procedure: The eluted fractions from the Ni-affinity Histrap columns were loaded on the gel filtration column in GF buffer at 1.20 mL/min. Eluted proteins were collected in 2 mL fractions. Eluted fractions were 95% pure as judged by SDS-PAGE.

Extraction

Procedure
Lysis buffer: 50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, Complete protease inhibitors (1 tablet/50 mL). Frozen cell pellets were thawed on ice over night and re-suspended in a total volume of 50 mL lysis buffer. The cells were disrupted by a high pressure cell disrupter followed by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI from a 5% (w/v) stock. After an incubation time of 15 minutes the lysate was centrifuged clear at 40 000xg for 30 minutes. The supernatant was then further clarified by filtration (0.45µm).
Concentration:Centricon with a 10kDa cut off in SEC-buffer.
Ligand
MassSpec:
Crystallization:Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/ml by mixing 100 nl of the concentrated protein with 100nl of a well solution containing 2M Potassium citrate. Plate like crystals appeared after 3 days at 4°C.
NMR Spectroscopy:
Data Collection:Crystals were cryo-protected using the well solution and 30% sucrose and flash frozen in liquid nitrogen. Diffraction data were collected at the SLS beam line X10 at a single wavelength (0.99 nm).
Data Processing:

References

Chiu YL , Coronel E, Ho CK, Shuman S, Rana TM. (2001) HIV-1 Tat protein interacts with mammalian capping enzyme and stimulates capping of TAR RNA. J Biol Chem. 276, 12959-12966.

Changela A , Ho CK, Martins A, Shuman S, Mondragon A (2001). Structure and mechanism of the RNA triphosphatase component of mammalian mRNA capping enzyme. EMBO J. 20, 2575-2586.

Meigang Gu and Christopher D Lima (2005) Processing the message: structural insights into capping and decapping mRNA. Current Opinion in Structural Biology 15, 99â€“106.