# Structure and Function of Essential Nucleoprotein ComplexesAlong a Viral Genome Packaging Pathway

> **NIH NIH R01** · UNIVERSITY OF COLORADO DENVER · 2020 · $370,006

## Abstract

Project Summary.
Bacteriophages play a major role in bacterial evolution, in mediating bacterial pathogenicity and antibiotic
resistance, in modulating the human microbiome and they have great potential as nanotherapeutics.
Understanding these issues with respect to human disease and harnessing their potential as theranostic
agents requires a fundamental understanding of virus development. The genome packaging pathways
are strongly conserved in the large double-stranded DNA (dsDNA) viruses, both prokaryotic and
eukaryotic. In this broad class of viruses, a terminase enzyme is responsible for (i) excision of an
individual genome from concatemeric substrate (genome maturation) and (ii) translocation of DNA into
a procapsid shell (genome packaging). These functions are catalyzed by terminase enzymes
assembled into discrete maturation and packaging motor complexes. Terminases are composed of a
catalytic subunit and a DNA recognition subunit, both of which are essential for genome packaging in
vivo. Structural and single-molecule studies have provided insight into packaging motor complexes
composed of the catalytic subunit in isolation; however, there is little information on motor complexes
containing both essential subunits. Further, there is a dearth of structural information on the equally
essential maturation complex precursor. This is due, in part, to the absence of well-characterized
holoenzyme preparations and a dearth of in vitro assays to comprehensively assess the pathway. We
have developed rigorous assays in which the biochemical, biophysical and structural features of the
lambda genome-packaging pathway can be defined in great detail. Using these tools, we propose to
characterize the structural (cryo-electron microscopy) and functional (biophysical, kinetic) features of the
maturation complex, which show mechanistic similarity to the tetrameric type IIE/F restriction
endonucleases. We directly address an emerging controversy relating to the DNA architecture in the
maturation complex that mediates complex stability. We next test the hypothesis that the lambda motor
also functions as a tetrameric complex and that ATP hydrolysis by the motor is strongly cooperative;
these features represent a significant departure from currently accepted paradigms. Finally, we
characterize a putative "nucleotide switch" mechanism that controls the transition from the stable
maturation complex to the dynamic motor complex bound to the capsid and we rigorously define the
energy budget of the translocating motor. The proposed studies will provide structural and mechanistic
detail on two sequential packaging complexes and their transition through the genome-packaging
pathway. These features are shared by all of the dsDNA viruses that package genomes from
concatemeric precursors (phage, herpes) and the results will be of broad and general significance.

## Key facts

- **NIH application ID:** 9920164
- **Project number:** 5R01GM127365-03
- **Recipient organization:** UNIVERSITY OF COLORADO DENVER
- **Principal Investigator:** Carlos Enrique Catalano
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $370,006
- **Award type:** 5
- **Project period:** 2018-05-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9920164

## Citation

> US National Institutes of Health, RePORTER application 9920164, Structure and Function of Essential Nucleoprotein ComplexesAlong a Viral Genome Packaging Pathway (5R01GM127365-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9920164. Licensed CC0.

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