Early events in the in Vitro packaging of bacteriophage λ DNA

Becker, Andrew; Marko, M A; Gold, Marvin

doi:10.1016/0042-6822(77)90100-3

Cited by 118 publications

(77 citation statements)

References 6 publications

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“…During the packaging of DNA, mature monomers are cleaved from the concatemer. The formation of precursors for DNA translocation, ternary complexes containing the prohead, the packaging enzyme, and the DNA, depends upon either ATP as an allosteric effector (T3, Shibata et al 1987) or ATP hydrolysis (l, Becker et al 1977;f-29, Guo et al 1987a). DNA translocation into the prohead is driven by ATP hydrolysis.…”

Section: Dna Packaging Machinerymentioning

confidence: 99%

“…Six copies of the 174 base-long prohead RNA (pRNA) are attached to the connector; pRNA is essential for binding of gp16 to the prohead (Guo et al 1987a). For l terminase, hetero-oligomers of gpNu1 (the small subunit) and gpA (the large subunit) bind to DNA to form a complex, called complex I, which then binds a prohead to form complex II, followed by DNA translocation (Becker et al 1977).…”

Section: Dna Condensation-dna Translocationmentioning

confidence: 99%

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Phage DNA packaging

1997

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Phage DNA packaging occurs by DNA translocation into a preformed protein shell-a prohead-with the aid of a packaging enzyme or a terminase. The packaging enzyme is composed of two subunits: the large subunit has ATP-binding, prohead binding, and DNA cleavage activities, and the small subunit is a DNA binding protein. DNA translocation is driven by ATP hydrolysis. In general, phage DNA replication mechanisms lead to the accumulation of concatemers. Concatemers are processed to mature DNA during and depending upon DNA packaging. This review will focus on the molecular mechanism of concatemer processing and the coupling of ATP hydrolysis to DNA translocation.

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Section: Dna Packaging Machinerymentioning

confidence: 99%

Section: Dna Condensation-dna Translocationmentioning

confidence: 99%

Phage DNA packaging

1997

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“…Complex I then binds a procapsid to generate complex II, a reaction which appears to be strongly enhanced by the product of the FI gene (Becker et al, 1977;Davidson and Gold, 1987).…”

Section: Introductionmentioning

confidence: 99%

Mutations of the coat protein gene of bacteriophage λ that overcome the necessity for the FI gene; the EFi domain

Murialdo

Tzamtzis

1997

Molecular Microbiology

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SummaryThe functions of most of the 10 genes involved in phage capsid morphogenesis are well understood. The function of the FI gene is one of the exceptions. Mutants in FI fail to mature and package DNA. The gene product (gpFI) seems to act as a catalyst for the formation of an intermediate in capsid assembly called complex II, which contains a procapsid (an empty capsid precursor), terminase (the enzyme that cleaves the DNA precursor and packages it into the procapsid) and DNA. The mechanism for this stimulation remains unknown. It has also been reported that gpFI appeared to stimulate terminase-mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. In comparison with other head-gene mutants, FI mutants are leaky, producing approx. 0.1 phage per infected cell. Some second-site revertants of FI ¹ phages, called 'fin', that bypass the necessity for gpFI, have been isolated and found to harbour a mutation in the genes that code for the two subunits of terminase. In the course of mapping additional fin mutants, it was discovered that some mapped outside the terminase genes. To localize the mutations, restriction fragments of fin mutant DNAs were subcloned into plasmids and their ability to contribute to fin function was determined by marker-rescue analysis. The location of the fin mutation was further delineated by deletion analysis of a plasmid that was positive for fin. This showed that some fin mutations mapped to a region comprising genes E, D and a portion of C. The sequencing of this entire region in several fin isolates showed that the fin mutations are clustered in a small region of gene E corresponding to a portion of 26 amino acid residues of the coat protein (gpE ). We have called this region of the protein the EFi domain.All the mutations result in an increase in positive charge relative to the wild-type protein. These results suggest that DNA maturation and packaging are in part controlled by an interaction between gpFI and capsid gpE.

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“…First, in a bulk reaction, we tethered biotinylated λ DNA fragments containing a cos packaging initiation site to streptavidin-coated microspheres. We then assembled the terminase complex onto the DNA by adding an extract containing recombinant gpA and gpNu1 proteins, which form a stable packaging intermediate referred to as Complex I 5,11,16,17 . We attached empty λ procapsids to separate microspheres coated with anti-λ procapsid antibodies.…”

Section: Initiation Of Single Dna Molecule Packagingmentioning

confidence: 99%

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

Fuller

Raymer

Rickgauer

et al. 2007

Journal of Molecular Biology

158

204

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Molecular motors drive genome packaging into preformed procapsids in many dsDNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage λ. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage ϕ29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged ~600 bp/s, which is 3.5-fold higher than ϕ29, and the motor processivity was also 3-fold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging in many cases, and greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid.

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Early events in the in Vitro packaging of bacteriophage λ DNA

Cited by 118 publications

References 6 publications

Phage DNA packaging

Phage DNA packaging

Mutations of the coat protein gene of bacteriophage λ that overcome the necessity for the FI gene; the EFi domain

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

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