The DNA polymerase of bacteriophage YerA41 replicates its T-modified DNA in a primer-independent manner

Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 angstrom in other phages, spacings of 33-36 angstrom between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome.Peer reviewe

The CRYO-EM structure of RNA polymerase I stalled at UV light-induced damage unravels a new molecular mechanism to identify lesions on ribosomal DNA

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-10-2019In eukaryotic cells, three RNA polymerases transcribe the genome, each specialized in transcribing a specific set of genes. Pol II synthesizes mRNA, Pol III produces short untranslated RNAs and Pol I transcribes ribosomal DNA (rDNA). The latter produces the rRNA precursor, which after maturation constitutes the backbone of the ribosome. Pol I accounts for approximately 60% of the total transcriptional activity in growing cells and also carries out the supervision of rDNA integrity. Therefore, it is a key determinant for the control of the normal function of the cell. Environmental threats can generate DNA lesions that are cytotoxic for the cell and one of the most known is UV-light. The principal DNA damage produced by this external agent is cis-syn cyclobutane pyrimidine dimers (CPDs), a bulky DNA lesion that can introduce distortions in the DNA helix, thus obstructing fundamental processes such as transcription. The main goal of this Ph.D. Thesis is understanding the structural basis of Pol I stalled at UV light-induced DNA damage. The principal contribution is the cryo-EM structure at 3.6 Å resolution and the derived atomic model of Pol I in elongation complex containing a CPD lesion at the DNA TS. This structure shows that the CPD lesion induces an early translocation intermediate, along with several conformational rearrangements in Pol I structural elements inside the DNA binding cleft, which contribute to enzyme stalling. The structure revealed that the BH residue R1015 plays a relevant role for enzyme arresting, which was confirmed by mutational analysis using E.coli RNA polymerase as a model system. In vitro transcription assays comparing the Pol I and Pol II behavior in the presence of CPD reveal that, while Pol II can slowly bypass the lesion, Pol I stalls right before the lesion due to the balance between a slow nucleotide incorporation and a fast-intrinsic RNA cleavage activity. Altogether, our results reveal the molecular mechanism of Pol I stalling at CPD lesions, which is distinct from Pol II arrest. This PhD Thesis opens the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNATesis realizada gracias a la ayuda BES-2014-070708 del Ministerio de Ciencia, Innovación y Universidade

The CRYO-EM structure of RNA polymerase I stalled at UV light-induced damage unravels a new molecular mechanism to identify lesions on ribosomal DNA

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 25-10-2019In eukaryotic cells, three RNA polymerases transcribe the genome, each specialized in transcribing a specific set of genes. Pol II synthesizes mRNA, Pol III produces short untranslated RNAs and Pol I transcribes ribosomal DNA (rDNA). The latter produces the rRNA precursor, which after maturation constitutes the backbone of the ribosome. Pol I accounts for approximately 60% of the total transcriptional activity in growing cells and also carries out the supervision of rDNA integrity. Therefore, it is a key determinant for the control of the normal function of the cell. Environmental threats can generate DNA lesions that are cytotoxic for the cell and one of the most known is UV-light. The principal DNA damage produced by this external agent is cis-syn cyclobutane pyrimidine dimers (CPDs), a bulky DNA lesion that can introduce distortions in the DNA helix, thus obstructing fundamental processes such as transcription. The main goal of this Ph.D. Thesis is understanding the structural basis of Pol I stalled at UV light-induced DNA damage. The principal contribution is the cryo-EM structure at 3.6 Å resolution and the derived atomic model of Pol I in elongation complex containing a CPD lesion at the DNA TS. This structure shows that the CPD lesion induces an early translocation intermediate, along with several conformational rearrangements in Pol I structural elements inside the DNA binding cleft, which contribute to enzyme stalling. The structure revealed that the BH residue R1015 plays a relevant role for enzyme arresting, which was confirmed by mutational analysis using E.coli RNA polymerase as a model system. In vitro transcription assays comparing the Pol I and Pol II behavior in the presence of CPD reveal that, while Pol II can slowly bypass the lesion, Pol I stalls right before the lesion due to the balance between a slow nucleotide incorporation and a fast-intrinsic RNA cleavage activity. Altogether, our results reveal the molecular mechanism of Pol I stalling at CPD lesions, which is distinct from Pol II arrest. This PhD Thesis opens the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNATesis realizada gracias a la ayuda BES-2014-070708 del Ministerio de Ciencia, Innovación y Universidade

The DNA polymerase of bacteriophage YerA41 replicates its T-modified DNA in a primer-independent manner

Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 angstrom in other phages, spacings of 33-36 angstrom between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome

Mechanism of Action of Group II Chaperonins:

Chaperonins are highly allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber which transiently provides a protected environment for protein folding. During evolution, two distinct strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. The present work contributes to the current mechanistical understanding of group II chaperonins by unraveling key functions of the built-in lid. In addition to physically encapsulating the substrate, the lid-forming apical protrusions also play a key role in regulating chaperonin function and ensuring its activity as a “two-stroke” molecular machine. By comparative investigation of two distinct chaperonin systems, namely TRiC and Mm-Cpn, this study uncovers a remarkable degree of mechanistic and functional conservation between group II chaperonins from eukaryotic and archaeal origin, despite their evolutionary distance

Summer Research Fellowship Project Descriptions 2022

A summary of research done by Smith College’s 2021 Summer Research Fellowship (SURF) Program participants. Ever since its 1967 start, SURF has been a cornerstone of Smith’s science education. Supervised by faculty mentor-advisors drawn from the Clark Science Center and connected to its eighteen science, mathematics, and engineering departments and programs and associated centers and units. At summer’s end, SURF participants were asked to summarize their research experiences for this publication.https://scholarworks.smith.edu/clark_womeninscience/1012/thumbnail.jp

The hunt for quasi-periodicities with wavelet and camera

Includes abstract. Includes bibliographical references (p. 357-379)

Structural studies of eukaryotic ribosome biogenesis and the sec and Bcs1 protein translocation systems

Three publications of this cumulative dissertation use cryo-electron microscopy (cryo-EM) to dissect the assembly pathway of the eukaryotic large ribosomal subunit (LSU). This pathway commences with freshly transcribed and initially unfolded rRNA in the nucleolus, which folds and incorporates ribosomal proteins while traveling to the cytoplasm, ultimately culminating in the mature LSU. During this highly complex pathway, the yeast cell must assemble four rRNAs and 79 ribosomal proteins with the help of over 200 assembly factors (AFs). Using cryo-EM, structures of nucleo\-plasmic and cytoplasmic assembly intermediates of the LSU could be solved in recent years, thus shedding light on the later stages of LSU formation. Early assembly steps remain enigmatic, as nucleolar LSU assembly intermediates have been biochemically but not structurally characterized. Taken together, we solved the structure of seven nucleolar or early nucleoplasmic intermediates at resolutions ranging from 3.3 to 4.5 Å, showing a linear assembly sequence. The first five structures show how the rRNA of the LSU is incorporated stepwise, in a non-transcriptional sequence, first forming the solvent exposed back side, and later the peptide exit tunnel and parts of the intersubunit surface (ISS). At the late nucleolar stage, the L1-stalk rRNA of domain V blocks the site of central protuberance (CP) assembly and is stabilized in a premature conformation by a range of AFs associated with the meandering, long N-terminal tail of Erb1. Two further structures show progression from this stage after release of the Erb1-Ytm1 complex by the Rea1 remodeling machinery. These intermediates, purified via Nop53, show dissociation of many early AFs from the premature ISS and destabilization of the L1-stalk. After subsequent release of the Spb1 methyltransferase, the L1-stalk rRNA can be accommodated in its mature conformation. This allows the premature CP to form, leading to a previously characterized nucleoplasmic intermediate, with a formed but premature CP. This particle is the substrate for the second Rea1 mediated structural remodeling, an intermediate of which we resolved to molecular resolution revealing Ipi1 as a central integrator for the Rix1-Ipi1-Ipi3 complex on this pre-60S particle. The binding of the Rix1-Rea1 remodeling machinery at this nucleoplasmic stage progresses maturation by inducing a 180$^{\circ}$ rotation of the 5S ribonucleoprotein particle (5S RNP) and CP. Using a combination of yeast genetics and cryo-EM we investigated the function of the AF Cgr1 in this maturation step. We showed that Cgr1 is required for CP rotation to take place, likely by stabilizing the rotated conformation. The Cgr1 function can be bypassed by introducing suppressor mutations in Rpf2 and Rrs1, two factors stabilizing the CP prior to rotation. Apart from ribosome biogenesis, two additional publications of this dissertation address protein translocation machinery, required for transport of proteins across or into membranes. The Sec translocon allows co- and posttranslational translocation of mostly unfolded substrates across the bacterial plasma and the eukaryotic endoplasmic reticulum (ER) membrane. We solved the structure of a stalled 70S ribosome-nascent chain complex (RNC) bound to the SecYEG translocon in a native like environment provided by a large lipid nanodisc. The structure shows all three subunits of the bacterial SecYEG complex and displays the lateral gate at a defined, early stage of opening or unzipping on the cytoplasmic side upon insertion of the signal anchor domain of the nascent chain. Specific pathways, such as the assembly of the mitochondrial bc1 respiratory chain complex, require folding of proteins in one compartment before translocation across a membrane to allow the protein to be active in another compartment. The bc1-complex component Rip1 folds in the mitochondrial matrix and assembles a 2Fe-2S cluster before being translocated across the inner mitochondrial membrane (IM) by the AAA-protein Bcs1. We solved the structure of Bcs1 in an ADP-bound state and two apo states, displaying a heptameric ring of Bcs1 protomers. Bcs1 forms two large aqueous vestibules separated by a seal forming middle domain. One vestibule is accessible from the matrix side and one lies within the inner mitochondrial membrane. The architecture and structural dynamics between the three states suggest an airlock like mechanism, allowing transport of folded Rip1 while maintaining the permeability barrier of the membrane

MOLECULAR AND ECOLOGICAL ASPECTS OF THE INTERACTIONS BETWEEN \u3ci\u3eAUREOCOCCUS ANOPHAGEFFERENS\u3c/i\u3e AND ITS GIANT VIRUS

Viruses are increasingly being recognized as an important biotic component of all ecosystems including agents that control the rapid ecological events that are harmful algal blooms (HABS). Aureococcus anophagefferens is a pelagophyte which causes recurrent ecosystem devastating brown tide blooms along the east coast of the USA and has recently spread to China and South Africa. It has been suggested that a large virus (AaV) is possibly an important agent for demise of brown tide blooms. This observation is consistent with the recognition of a number of other giant viruses modulating algal blooms in marine systems. In this dissertation, we investigated both the molecular underpinnings of Aureococcus-AaV interactions and the dynamics of AaV and the associated viral community in situ. We determined the genome sequence and phylogenetic history of AaV using high throughput sequencing approach and revealed it’s intertwined evolutionary history with the host and other organisms. Building upon the available genome of AaV and its host, we took an RNA-seq approach to provide insights on the physiological state of the AaV-infected Aureococcus ‘virocell’ that is geared towards virus production. In situ activity of AaV was detected by targeted amplicon and high throughput community RNA sequencing (metatranscriptomics) from Quantuck Bay, NY, a site with recurrent brown tide blooms. AaV and associated giant algal viruses in the Mimiviridae clade were found to respond to environmental changes, indicating that this newly recognized phylogenetic group is an important contributor to the eukaryotic phytoplankton dynamics. Analyzing time series metatranscriptomics from two distinct coastal sites recovered diverse viruses infecting microeukaryotes (including AaV) as part of interacting networks of viruses and microeukaryotes. Results from these studies testify AaV as an important factor for brown tide bloom demise, reveals the molecular underpinnings of AaV-host interactions and establishes the ecological relevance of Mimivirus-like algal viruses. We also provide foundation for using metatranscriptomics as an important tool in marine virus ecology – capable of recovering associations among coexisting marine microeukaryotes and viruses