DNA:n pakkaus ja isäntäsolun hajotus : Bakteriofaagi PRD1 -infektion loppuvaiheen tapahtumat

The object of this study is a tailless internal membrane-containing bacteriophage PRD1. It has a dsDNA genome with covalently bound terminal proteins required for replication. The uniqueness of the structure makes this phage a desirable object of research. PRD1 has been studied for some 30 years during which time a lot of information has accumulated on its structure and life-cycle. The two least characterised steps of the PRD1 life-cycle, the genome packaging and virus release are investigated here. PRD1 shares the main principles of virion assembly (DNA packaging in particular) and host cell lysis with other dsDNA bacteriophages. However, this phage has some fascinating individual peculiarities, such as DNA packaging into a membrane vesicle inside the capsid, absence of apparent portal protein, holin inhibitor and procapsid expansion. In the course of this study we have identified the components of the DNA packaging vertex of the capsid, and determined the function of protein P6 in packaging. We managed to purify the procapsids for an in vitro packaging system, optimise the reaction and significantly increase its efficiency. We developed a new method to determine DNA translocation and were able to quantify the efficiency and the rate of packaging. A model for PRD1 DNA packaging was also proposed. Another part of this study covers the lysis of the host cell. As other dsDNA bacteriophages PRD1 has been proposed to utilise a two-component lysis system. The existence of this lysis system in PRD1 has been proven by experiments using recombinant proteins and the multi-step nature of the lysis process has been established.DNA:n pakkaus ja isäntäsolun hajotus: Bakteriofaagi PRD1 -infektion loppuvaiheen tapahtumat Virukset ovat solunsisäisiä loisia, jotka käyttävät hyväkseen isäntäsolunsa resursseja tuottaakseen uusia viruskopioita. Tämän tutkimuksen kohteena on eräs bakteereja infektoiva virus, PRD1. PRD1:n rakenne on bakteerivirukseksi poikkeuksellinen, ja se on osittain samankaltainen adenoviruksen kanssa. Työssä on keskitytty PRD1:n elinkierron kahteen viimeiseen vaiheeseen: viruksen genomin pakkaamiseen ja isäntäsolun hajotukseen. Viruksen perimäaineksen, pitkän DNA- tai RNA-molekyylin, pakkaaminen tiukasti hyvin pieneen, suojaavaan proteiinikuoreen eli kapsidiin edelleen kuljetettavaksi seuraavaan kohteeseen on kiehtova prosessi. Tässä työssä on tutkittu PRD1:n perimäaineksen eli genomin pakkaamista kapsidiin. Kun viruksen genomi on monistettu solussa ja uudet viruspartikkelit on koottu, on niiden päästävä ulos solusta infektoimaan uusia isäntäsoluja. Tässä työssä on selvitetty, miten ja milloin PRD1-infektiossa isäntäsolu hajotetaan uusien viruksien vapauttamiseksi. Toivon mukaan tämä väitöskirjatyö voi omalta osaltaan tuoda lisävalaistusta käsityksiimme näistä bakteerivirusten perustavanlaatuisista prosesseista

Dual-trap optical tweezers with real-time force clamp control

Single molecule force clamp experiments are widely used to investigate how enzymes, molecular motors, and other molecular mechanisms work. We developed a dual-trap optical tweezers instrument with real-time (200 kHz update rate) force clamp control that can exert 0–100 pN forces on trapped beads. A model for force clamp experiments in the dumbbell-geometry is presented. We observe good agreement between predicted and observed power spectra of bead position and force fluctuations. The model can be used to predict and optimize the dynamics of real-time force clamp optical tweezers instruments. The results from a proof-of-principle experiment in which lambda exonuclease converts a double-stranded DNA tether, held at constant tension, into its single-stranded form, show that the developed instrument is suitable for experiments in single molecule biology.Peer reviewedPeer reviewe

Single-molecule measurements of viral ssRNA packaging

Genome packaging of double-stranded RNA (dsRNA) phages has been widely studied using biochemical and molecular biology methods. We adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. To our knowledge, this is the first attempt to study the details of viral RNA packaging using optical tweezers. Pseudomonas phage phi 6 is a dsRNA virus with a tripartite genome. Positive-sense (+) single-stranded RNA (ssRNA) genome precursors are packaged into a preformed procapsid (PC), where negative strands are synthesized. We present single-molecule measurements of the viral ssRNA packaging by the phi 6 PC. Our data show that packaging proceeds intermittently in slow and fast phases, which likely reflects differences in the unfolding of the RNA secondary structures of the ssRNA being packaged. Although the mean packaging velocity was relatively low (0.07-0.54 nm/sec), packaging could reach 4.62 nm/sec during the fast packaging phase.Peer reviewe

Efficient DNA Packaging of Bacteriophage PRD1 Requires the Unique Vertex Protein P6

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented