A. Gritsenko
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6 records found
1
In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles. Despite markedly condensing the bacterial chromosome, the nucleoid-structuring protein Dps selectively allows access by RNA polymerase and transcription factors at normal rates while excluding other factors such as restriction endonucleases.
Translation of mRNAs through Internal Ribosome Entry Sites (IRESs) has emerged as a prominent mechanism of cellular and viral initiation. It supports cap-independent translation of select cellular genes under normal conditions, and in conditions when cap-dependent translation is inhibited. IRES structure and sequence are believed to be involved in this process. However due to the small number of IRESs known, there have been no systematic investigations of the determinants of IRES activity. With the recent discovery of thousands of novel IRESs in human and viruses, the next challenge is to decipher the sequence determinants of IRES activity. We present the first in-depth computational analysis of a large body of IRESs, exploring RNA sequence features predictive of IRES activity. We identified predictive k-mer features resembling IRES trans-acting factor (ITAF) binding motifs across human and viral IRESs, and found that their effect on expression depends on their sequence, number and position. Our results also suggest that the architecture of retroviral IRESs differs from that of other viruses, presumably due to their exposure to the nuclear environment. Finally, we measured IRES activity of synthetically designed sequences to confirm our prediction of increasing activity as a function of the number of short IRES elements.
Data-driven modelling of protein synthesis
A sequence perspective
Second generation DNA sequencing technologies have made it easy and affordable to reconstruct the genomes of industrially relevant microbes, thus providing better reference sequences for genetic engineering. However, technological limitations allow for reconstructing only parts of the entire genomes unambiguously, thus requiring additional scaffolding steps to obtain genome-length reconstructions. We propose a method that improves genome scaffolding by integrating heterogeneous sources of information on genome contiguity. These methods improve the quality of genome reconstructions at the cost of a limited number of additional errors.
The ease and affordability of DNA sequencing has also led to the development of a number of biological assays which exploit sequencing, among which the ribosome profiling assay. This assay allows for unprecedented examination of the process of protein synthesis by recording positions of actively translating ribosomes across thousands of living cells. We employed these data to develop data-driven models of Saccharomyces cerevisiae protein synthesis. A relatively simple model was used to re-design genes for heterologous expression; a second, more complex model yielded insights into the process of translation. Our models suggest that protein synthesis is limited at the stage of initiation, and that codon translation rates are not determined by tRNA levels alone, and appear to be sequence context-dependent.
Finally, the combination of DNA synthesis and sequencing offers the possibility to perform high-throughput in vivo assays to study the effect of user-designed sequences. We used this approach to study translation initiation at Internal Ribosome Entry Sites (IRESs). We identified short sequence elements predictive of IRES activity in viruses and humans, and obtained insights into the effect of element sequence, multiplicity and position on IRES activity. We propose a high-level architecture of viral and cellular IRESs, and offer a mechanistic explanation for differences between IRES architectures of different virus types. ...
Second generation DNA sequencing technologies have made it easy and affordable to reconstruct the genomes of industrially relevant microbes, thus providing better reference sequences for genetic engineering. However, technological limitations allow for reconstructing only parts of the entire genomes unambiguously, thus requiring additional scaffolding steps to obtain genome-length reconstructions. We propose a method that improves genome scaffolding by integrating heterogeneous sources of information on genome contiguity. These methods improve the quality of genome reconstructions at the cost of a limited number of additional errors.
The ease and affordability of DNA sequencing has also led to the development of a number of biological assays which exploit sequencing, among which the ribosome profiling assay. This assay allows for unprecedented examination of the process of protein synthesis by recording positions of actively translating ribosomes across thousands of living cells. We employed these data to develop data-driven models of Saccharomyces cerevisiae protein synthesis. A relatively simple model was used to re-design genes for heterologous expression; a second, more complex model yielded insights into the process of translation. Our models suggest that protein synthesis is limited at the stage of initiation, and that codon translation rates are not determined by tRNA levels alone, and appear to be sequence context-dependent.
Finally, the combination of DNA synthesis and sequencing offers the possibility to perform high-throughput in vivo assays to study the effect of user-designed sequences. We used this approach to study translation initiation at Internal Ribosome Entry Sites (IRESs). We identified short sequence elements predictive of IRES activity in viruses and humans, and obtained insights into the effect of element sequence, multiplicity and position on IRES activity. We propose a high-level architecture of viral and cellular IRESs, and offer a mechanistic explanation for differences between IRES architectures of different virus types.
Comparative genetics
Systematic discovery of cap-independent translation sequences in human and viral genomes
To investigate gene specificity at the level of translation in both the human genome and viruses, we devised a high-throughput bicistronic assay to quantify cap-independent translation.We uncovered thousands of novel cap-independent translation sequences, and we provide insights on the landscape of translational regulation in both humans and viruses.We find extensive translational elements in the 3? untranslated region of human transcripts and the polyprotein region of uncapped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity, we identify potential mechanisms of secondary structure, short sequence motif, and base pairing with the 18S ribosomal RNA (rRNA). Furthermore, we systematically map the 18S rRNA regions for which reverse complementarity enhances translation. Thus, we make available insights into the mechanisms of translational control in humans and viruses.