Mechanism of Stimulation of the

DNA-packaging ATPase in Bacteriophage T4

Karoly Viragh

Comprehensive Seminar

March 13, 2003

1.1. IntroductionIntroduction• Bacteriophage T4 DNA packagingBacteriophage T4 DNA packaging• Arginine-finger hypothesisArginine-finger hypothesis

2.2. Experimental DesignExperimental Design• Peptide design and synthesisPeptide design and synthesis• Preliminary evaluation of ATPase activityPreliminary evaluation of ATPase activity

3.3. Results and DiscussionResults and Discussion• PCR-amplification and purification of coding DNAPCR-amplification and purification of coding DNA• CloningCloning• Peptide Expression and PurificationPeptide Expression and Purification• ATPase assayATPase assay

4.4. ConclusionsConclusions5.5. QuestionsQuestions

Overview

Intro – Bacteriophage T4

Viruses - obligate parasites, which depend on the host cells that they infect to reproduce

Bacteriophages - viruses that infect bacteria

T4 - specifically infects E. coli

Negative Contrast Electron Micrograph of T4

Head Assembly of Bacteriophage T4

T4 DNA packaging

The process of moving the 170 kbp genomic dsDNA into the empty capsid and its subsequent organization

T4 Packaging Machine:

• gp20 (61 kDa structural component – portal vertex)

• gp17 (70 kDa large terminase/packaging protein)

• gp16 (18 kDa small terminase/packaging protein)

Intro – T4 DNA packaging

Arginine finger(s) in Ras and G-proteins

Recent analyses of G-protein GTPases suggest that the

catalytic stimulation of GTP-hydrolysis is due to a precise

positioning of one or more arginine residues (“arginine fingers”) of a GAP (GTPase Activating Protein) into the GTPase catalytic center

Intro – GTPase catalytic center

 

T4: M-----EGLDINKLLDISDLPG-IDGEEIKV-YEPLQLVEVKSNPQNRTPDLEDDYGVVRRNMHFQQQMLMDAAK 68

RB49: M--------KQLMNFESLGLPGSFDAEEDRVSYDPLVLTPVESHPEDRNIDLQRDYNEARQNIHFQNQMMMDAAK 67

KVP40: MNDELMQQLQALTQVDGLDLPGAIEAPEPEE-FQPPVIKEVESHPTERVKDLEADYATVRDNAHFQQQLLRMAAL 74

KVP20: MNDELMKQLQALTQVDELDLPGAIEAEEPEE-FQPPVIKEVESHPSERVKDLESDYATVRDNAHFQQQLLRMAAM 74

Con1: M--------------+---LPG--+--E----+-P--+--V+S+P--R--DL+-DY---R-N-HFQ-Q++--AA-

 

T4: IFLETAKNADSPRHMEVFATLMGQMTTTNREILKLHKDMKDITSEQ-VG--TKGAVPTGQMNIQNATVFMGSPTE 140

RB49: IYLELAKNSESPRFLQAFNGLMQQMSNNNKELLNIHKDMKKIT-EQ-AGEKKKDNTPAAPVNIQNATVFMGSPSD 139

KVP40: KAFENASMSDAPRMMEVFATLMTQMTNNNKQILDIQKQMKDITQQEIASPQGGGGGTVQSINAETA-VFVGNARD 148

KVP20: KAFENASMSDAPRMMEVFATLMTQMTNNNKQILDIQKQMKDITQQEIATAQGGSGGTVQSINAETA-VFVGNSRD 148

Con1: ---E-A--+++PR-++-F--LM-QM+--N+++L-+-K-MK-IT-++--------------+N-+-A-VF+G+--+

 

T4: LMDEIGD-----------AYEAQEAREKVINGTTD 164

RB49: LMDEIED---------------EEAR--VIEGETV 158

KVP40: LLNEVGSRQEYLRNKKEEEIIDVEPEEKVQEKDD- 182

KVP20: LLNEVGSRQEYLRSKREEEIIDVEPEEKVQEKDD- 182

Con1: L++E+------------------E----V------

Sequence analysis of the gp16 subunit of four bacteriophages in the T4 family identified strictly conserved arginine residues: R41, R53 and R81

Intro – Hypothesis

Formation of the gp16-gp17 enzyme complex

1. allows the positioning of one or more arginine fingers from gp16 into the gp17 ATPase catalytic center, which

2. stimulates ATPase and DNA packaging activities by stabilizing the transition state for ATP hydrolysis

Experimental Design

A. Site-directed mutagenesis (not used)

B. Peptide-modeling approach (employed here)

1. Design truncated gp16-peptides based on 2° structure predictions by a variety of online computational tools

2. Synthesize peptides using a molecular genetic approach (Peptide 2)

a. PCR-amplify the gene coding for Peptide 2

b. Purify the amplified DNA fragment

c. Clone the DNA fragment into a plasmid vector

d. Induce the expression of Peptide 2 from the vector

e. Purify Peptide 2 using Ni2+-Agarose chromatography

3. Evaluate ATPase activity (preliminary results)

The predicted secondary structures for gp16, as well as the two peptides: Peptide 1 (68 amino acids from 27 to 94) and Peptide 2 (73 amino acids from 47 to 119) [H=alpha-helix, E=extended(beta)-sheet, –= random coil, T=beta-turn, L=omega-loop]. 1 2 3 4 5 6 MEGLDINKLLDISDLPGIDGEEIKVYEPLQLVEVKSNPQNRTPDLEDDYGVVRRNMHFQQ 0. ------------------------------E-------------HHHHHHHHHHHHHHHH 1. -----H----------------E------HHEE-----------------HH---HHHHH 2. LLL---L-----LLLLLLLL-EEEEE---EEEEE-LLLLLLLL-LLLL---------HHH 3. HTT----HEE-----TT-----EEEE--HEEEEE-------------HHHEEHHHHHHHH 4. ---------------------EE------EEEEE---------------HHHHHHHHHHH 5. -----HHHHH-----------EEEE----EEEEE-------------HHHHHHH--HHHH 6. ---HHHHHHHH---------HHHHHHHHHHHHHHH-T-------HH----EEHHHHHHHH 7 8 9 0 1 2 QMLMDAAKIFLETAKNADSPRHMEVFATLMGQMTTTNREILKLHKDMKDITSEQVGTKGA 0. HHHHHHHHHHHHH-------HHHHHHHHHHHHHHH-HHHHHHHH----HHHHHHH----- 1. HHHHHHHHHHHHHH--------HHHHHHHH-------HHHHHHH---------E------ 2. HHHHHHHHHHHHHH---LL-HHHHHHH-HH------HHHHHHHH---------E------ 3. HHHHHHHHHHHHH--------HHHHHHHHHHHH---HHHHHHHHHHHHHHHHH-ETTT-- 4. HHHHHHHHHHHHHH-------HHHHHHHHHHHHHHHHHHHHHHHH----HHHHH------ 5. HHHHHHHHHHHHH-------HHHHHHHHHH----HHHHHHHHHHHHHHHH---------s- 6. HHHHHHHHHHHHHHHHH---HHHHHHHHHHHHHH--HHHHHHHHHHHHHH-HHHHT---- 3 4 5 6 VPTGQMNIQNATVFMGSPTELMDEIGDAYEAQEAREKVINGTTD (164 amino acids) 0. -----EEE--EEEEE----HHHHHHHHHHHHH---EEEE----- 1. -----EE----EEE-------HHHH---HHHHHHHHHEE----- 2. --L---E----EEE-LL-HHHHHHHHHHHHHHHHHHHHH-LLLL 3. --------TT-EEEE---HHHHHHHHHHHHHHHHHHHHHHT--- 4. ---------EEEEE-----HHHHHHHHHHHHHHHHHHHEEEEE- 5. -----EEE---EEE---HHHHHHHHHHHHHHHHHHHHHH----- 6. ----HH-HHHHHEHH----HHHHHHHHHHHHHHHHHHHH-----

Results – PCR AmplificationPCR amplification of the peptide-coding regions from T4 genomic DNA (P1=Peptide 1, P2=Peptide 2)

Results – DNA purificationAgarose gel purification of the DNA fragment coding for Peptide 2 (P2)

Results – CloningTesting for the presence of the right insert in the correct orientation by PCR

Results – InductionSDS-PAGE analysis of IPTG-induced protein expression

Results – Peptide PurificationSDS-PAGE analysis of the purification process for Peptide 2

Results – ATPase assayATPase assay to evaluate the activity of the truncated gp16 protein (Peptide 2)

Conclusions

1. T4 DNA packaging is a complex process involving the small terminase subunit gp16.

2. The critical segments of gp16 can be evaluated using the peptide modeling approach.

3. Peptide 2 does not stimulate gp17 ATPase activity.

4. More complete biochemical analysis is needed.

Acknowledgements

I’d like to thank:

• Dr. Rao, for his invaluable tutelage throughout the experiments;

• Dr. Kovach, for her assistance in revising the report;

• Dr. Kondabagil, for his help in protein purification and ATPase assays

On a final note…

This presentation and the comprehensive paper are available on-line at

http://www.karoly.tk

Any questions?

Thank you for your time!