Category:Team Mu

Figure 9

Comparing the portal position in the procapsid and the virion shows that the portal increases its contact with the capsid shell during maturation (Figure S1). We propose that this portion of the scaffold remains in place during dsDNA packaging, allowing access of the gp4 C terminus to the bottom of the portal. When gp4 binds, the scaffold protein is displaced allowing the final conformational change implied by the position of the gp4-C-terminal polypeptide that is wedged between the capsid and portal.

Fro. I. Dependence of phage formation in vitro on the concentration of the capsid donor extract.

Infected cell[ extracts containing incomplete capsids were freshly thawed and lysed as described in Materials and Methods. Diluted .samples were mixed with concentrated protein donor extract {5-) and incubated for I h at 23°C, at which time the reactions were stopped by further dilution and titered for viable phage. The background of the protein donor extract was 102 phage/mi. The background of the • at mo.~l, Conc-entrated calmid donor extracts were (0) 3.3× 10 (4- extract), (t) 2:5 × l0 T (10- extract) and (at) 2 x 103 (26-extract).

Dependence of in vitro assembly on the concentration of the 4", lO ÷ and 26* activities. (a)

Complementation of 10- extracts (O), 26- extracts (11) and 4- extracts (~lk) with protein donor extract. The capsid extracts were diluted 25-fold, to a final infected cell conventration of about 2 x 10 ° c~lls/ml. Samples were mixed with serial dilutions of the 5- protein donor. The maximum slo|~s determined from these curve,a were: 4+ activity, 5 to 6; 26 + activity. I to 2; 10 + activity, 4 to 5. The background of the undiluted extracts were protein donor 10a; 4- extract < 10s; 10- extracts, 5 x 10~; 26- extract, 2 x l0 a. (b) Variation of the ratio of cap`aid,a to protein; complemcntation of 4- capsids with cap,aid donor cell concentration: (O) 5x lO 9 cell,a/ml; (A) 2 x l0 s cell,a/ml; (m) 4 x 10 v (~lls/ml. The protein donor extract was from the same batch a.s used in (a).

Fro. 4. Dependence of in vitro a.~.~em!~ly on the concentration of the 4", lO ÷ and 26* activities. (a)

Com|)lementation of 10- extracts (O), 26- extracts (11) and 4- extracts (~lk) with protein donor extract. The cap,aid extracts were diluted 25-fold, to a final infected cell cont'entration of about 2 x 10 ° c~lls/ml. Samples were mixed with serial dilutions of the 5- prc)tein donor. The maximum slo|~s determined from these curve,a were: 4+ activity, 5 to 6; 26 + activity. I to 2; 10 + activity, 4 to 5. The background of the undiluted extracts were protein donor 10a; 4- extract < 10s; 10- extracts, 5 x 10~; 26- extract, 2 x l0 a. (b) Variation of the ratio of cap`aid,a to protein; complemcntation of 4- ~'apsids with cap,aid donor cell concentration: (O) 5x lO 9 cell,a/ml; (A) 2 x l0 s cell,a/ml; (m) 4 x i0 v (~lls/ml. The protein donor extract was from the same batch a.s used in (a).

ependenc~ of phage formation in vitro on the concentration of the capsid donor extract.

Infected ¢~zl[ extracts containing incomplete capsids were freshly thawed and lysed as described in Materials and Methods. D~uted .samples were mixed with concentrated protein donor extract {5-) and incubated for I h at 23°C, at which time the reactions were stopped by further dilution and titered for viable phage. The background of the protein donor extract was 102 phage/mi. The background of the • at mo.~l, Conc-entrated calmid donor extracts were (0) 3.3× 10 (4- extract), (t) 2:5 × l0 T (10- extract) and (at) 2 x 103 (26-extract).

Fro. I. Dependenc~ of phage formation in vitro on the concentration of the capsid donor extract.

Infected ¢~zl[ extracts containing incomplete capsids were freshly thawed and lysed as described in Materials and Methods. D~uted .samples were mixed with concentrated protein donor extract {5-) and incubated for I h at 23°C, at which time the reactions were stopped by further dilution and titered for viable phage. The background of the protein donor extract was 102 phage/mi. The background of the • at mo.~l, Conc-entrated calmid donor extracts were (0) 3.3× 10 (4- extract), (t) 2:5 × l0 T (10- extract) and (at) 2 x 103 (26-extract).

Fro. 4. Dependence of in vitro a.~.~em!~ly on the concentration of the 4", lO ÷ and 26* activities. (a)

Com|)lementation of 10- extracts (O), 26- extracts (11) and 4- extracts (~lk) with protein donor extract. The cap,aid extracts were diluted 25-fold, to a final infected cell cont'entration of about 2 x 10 ° c~lls/ml. Samples were mixed with serial dilutions of the 5- prc)tein donor. The maximum slo|~s determined from these curve,a were: 4+ activity, 5 to 6; 26 + activity. I to 2; 10 + activity, 4 to 5. The background of the undiluted extracts were protein donor 10a; 4- extract < 10s; 10- extracts, 5 x 10~; 26- extract, 2 x l0 a. (b) Variation of the ratio of cap`aid,a to protein; complemcntation of 4- ~'apsids with cap,aid donor cell concentration: (O) 5x lO 9 cell,a/ml; (A) 2 x l0 s cell,a/ml; (m) 4 x i0 v (~lls/ml. The protein donor extract was from the same batch a.s used in (a).

Figure 7. Isolating the gp1:gp4 assembly intermediate on agarose gel. (a) Native agarose gel run at 30 °C showing a

stably populated gp(1)12:gp(4)6 assembly intermediate. In lane 1 is dodecameric portal protein gp(1)12. The gp(1)12:gp(4)6 assembly intermediate in lanes 2 and 3 migrates on gel as a slightly lower mobility band, clearly distinguishable from fully saturated decorated gp(1)12:gp(4)12 complex in lane 4 and free dodecameric portal protein gp(1)12 in lane 4. The intermediates in lanes 2 and 3 were formed by adding six equivalents of gp4 to gp(1)12 and incubating the complex at 30 °C for 30 s and 30 min, respectively. In lane 4 approximately eight equivalents of gp4 were added, yielding the fully decorated gp(1)12:gp(4)12 complex and free gp(1)12. (b) Titration of gp4 binding to gp1 at 30 °C. By running the agarose gel at 30 °C both gp(1)12:gp(4)6 assembly intermediate and fully decorated gp(1)12:gp(4)12 portal protein are visible on the same titration. The assembly intermediate appears at stoichiometries gp(1):gp(4) equal to 12 (lanes 3−6) and fades away in lane 7, where >six equivalents of gp4 are present.

Micrographs of the gp1:gp4

complex were compared to gp4-free portal rings, as shown in Figure 9(a) and (b), respectively. The majority of gp1:gp4 complexes seen on the micrographs displayed a preferential orientation on the grid with the central hole perpendicular to the grid. In rare instances single complexes and head-tohead dimers of the gp1:gp4 complex were seen in side view (see higher magnifications in Figure 9(b)). Such side views are particularly informative, in that the dodecameric portal protein without bound gp4 forms a mushroom-shaped structure with a channel through the center

Figure 9. Negative stain electron microscopy of tail accessory factor gp4 bound to P22 portal protein rings. (a)

Negative stain electron microscopy image of purified P22 portal protein rings. Many “donut-like” structures are visible, which in most (rarer) side view cases adopt a head-to-head conformation. (b) Negative stain micrograph of gel filtration purified portal protein in complex with gp4.

Micrographs of the gp1:gp4

complex were compared to gp4-free portal rings, as shown in Figure 9(a) and (b), respectively. The majority of gp1:gp4 complexes seen on the micrographs displayed a preferential orientation on the grid with the central hole perpendicular to the grid. In rare instances single complexes and head-tohead dimers of the gp1:gp4 complex were seen in side view (see higher magnifications in Figure 9(b))

Figure 10 shows that gene D protein, known to be present in the phage head, does not affect head size, suggesting it provides head stability instead. This is verified in Figure 11, which shows that gene D mutants are more susceptible to EDTA inactivation through the rupturing of the phage head. Protein D is likely a decorator protein as it stabilizes the head but is not essential in the gene D mutants isolated (Figure 2).

Gene 1.0 of phage T7 was cloned into and expressed on a plasmid vector. The protein produced from the expression of gene 1.0 was demonstrated to be a T7 RNA polymerase due to its ability to transcribe RNA from T7 DNA, when in the presence of rifampicin (T7 RNA polymerase is known to be unaffected by rifampicin, which is an antibiotic that inhibits bacterial RNA polymerase).

Fig 5. The T3 gene 1.0 was over-expressed on a plasmid, and the protein product was collected. T3 mutants with no functional gene 1.0 were incapable of initiating transcription, but regained function when in the presence of collected gp1.0 protein. T7 phages without a functional copy of gene 1.0 were not affected by phage T3 gp1.0. This suggests that the T3 gene 1.0 is a T3-specific RNA polymerase.

Figure 3: A nitrocellulose filter was used to determine levels of binding between gp2.5 and radioactively labeled DNA. Nitrocellulose is negatively charged, so only DNA bound to gp2.5 could be retained. With increasing concentrations of gp2.5, greater amounts of single-stranded DNA were retained. The experiment only demonstrated a small amount of binding between gp2.5 and double stranded DNA. This suggests that gp2.5's molecular function is the binding of ssDNA.

Figs 3,4

Table 2 shows that anti-H antibody inhibits infectivity of the phiX174 SS complex with gene H protein. Figure 3 and Table 3 demonstrate that the Gene H protein is recognized specifically by the phage LPS receptor previously identified. This is evidence implicating gene H in LPS-binding and phage adsorption.

Figure 2A: Researchers purified T7 virions from E.coli cells and separated the viral heads. Cryo-electron microscopy was used to examine the empty viral heads, and the structure of gp10A was determined to be that of a T=7 icosahedral capsid, major subunit.

Figure 4 shows results of a holin complementation assay. This gene was inserted with lysis cassettes missing the holin gene to confirm holin function. When inserted with the lambda cassette (lambda endolysin), abrupt lysis pattern was not seen. However, lysis is accelerated with expression of this holin gene and a SAR endolysin, suggesting pinholin activity rather than canonical holin activity.

Figure 1 pinpoints the major capsid protein through 2-dimensional gel electrophoresis. Figure 2 displays gel electrophoretic analysis of extracts produced by lysogens containing amber mutant prophages. T mutants were the only late gene mutants which lacked the major capsid protein, suggesting it is the T gene that encodes for this protein.

Incubation of MS2 coat protein with RNA showed significantly decreased phage protein production through 14C-labeled proteins and radioautography. The coat protein was shown to have specific inhibitory effect on its homologous RNA, and formed a complex when bound to the RNA. Fig. 5 shows that formation of this complex strongly correlates to inhibitory effect.

Fig 3

Fig 3