Project Details
Projekt
Systematic identification of novel µ-proteins in bacteria using ribosome profiling data
Applicant
Professorin Dr. Zoya Ignatova
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Term
from 2017 to 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 378478032
Emerging evidence places short proteins (µ proteins) more centrally in physiological processes. However, their de novo identification is difficult and they are often missed by algorithms searching for new open-reading frames because of the short size of the µ-proteins, often <150 nt or <50 codons. In this project, we seek to address the current void in algorithms/tools for systematic identification of short ORFs (sORFs) in bacteria which encode µ-proteins. We will use the power of two deep-sequencing technologies, RNA-Seq and ribosome profiling, to extract transcriptome-wide expression features of sORFs and use them in designing the algorithm for de novo search of expressed sORFs. These features include translational start, ribosome release score reporting on termination of protein synthesis, and most importantly, the three-nucleotide periodicity in ribosome-protected fragments when truly translating an ORF. To faithfully determine all initiation sites (including non-canonical starts), we will sequence the transcriptome upon inhibition of translation initiation with a newly identified peptide antibiotic. Since bacteria in general employ similar expression rules, it is our vision to train the algorithm with data sets produced in E. coli MG1655 grown at various conditions, and to develop it to work across all bacterial species. Leveraging expression features from RNA-Seq and ribosome profiling, which allow predicting only truly expressed sORFs, is a step forward in detecting sORFs as compared to the algorithms using solely genetic information.µ-proteins might be expressed only under certain growth or environmental conditions. Thus, to address their role in shaping stress response, we aim to probe sORF expression at both translation and transcription level at various stresses (heat, oxidative and osmotic stress) using ribosome profiling and RNA-Seq data sets. We will carry out further characterization of some of these newly discovered µ-proteins (preferably those that are expressed only under certain stress conditions) to elucidate expression on the protein level and interaction partners using genomic tagging for pulldown experiments.
DFG Programme
Priority Programmes
