Biology subjects

Ignatova, Z.

Publications and source records attributed to Ignatova, Z..

3 recordsLinked to original sources

bioRxiv2018

Structure of a hibernating 100S ribosome reveals an inactive conformation of the ribosomal protein S1

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles1. In {gamma}-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF)2-4. Although structures of E. coli 100S particles have been reported5,6, the low resolution (18-38 [A]) prevented the mechanism of ribosome inactivation and dimerization to be fully elucidated. Here we present single particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary phase E. coli cells at 3.0-7.9 [A] resolution, respectively. Preferred orientation bias for the complete 100S particle was overcome using tilting during data collection. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site tRNA and ribosomal protein S1 in the 100S particle. HPF interacts with the anticodon-stem-loop of the E-tRNA and occludes the binding site for the mRNA as well as A- and P-site tRNAs. RMF stabilizes a compact conformation of S1, which together sequester the anti-Shine-Dalgarno (SD) sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, S1 and S2 form intersubunit bridges with S3 and S4 and the C-terminus of S2 probes the mRNA entrance channel of the symmetry related particle, thus suggesting that only translationally inactive ribosomes are prone to dimerization. The back-to-back 100S dimerization mediated by HPF and RMF is distinct from that observed previously in Gram-positive bacteria7-10 and reveals a unique function for S1 in ribosome dimerization and inactivation, rather than its canonical role in facilitating translation initiation.

molecular biology↗

bioRxiv2018

Dynamic m6A methylation facilitates mRNA triaging to stress granules

Summary blurbm6A-modification in the 5 vicinity of the coding sequence of transcripts provides a selective mechanism for triaging mRNAs to stress granules and is mediated by YTHDF3 reader protein.\n\nAbstractReversible post-transcriptional modifications on messenger RNA emerge as prevalent phenomena in RNA metabolism. The most abundant among them is N6-methyladenosine (m6A) which is pivotal for RNA metabolism and function, its role in stress response remains elusive. We have discovered that in response to oxidative stress, transcripts are additionally m6A-modified in their 5 vicinity. Distinct from that of the translationally-active mRNAs, this methylation pattern provides a selective mechanism for triaging mRNAs from the translatable pool to stress-induced stress granules. These stress-induced newly methylated sites are selectively recognized by the YTH domain family 3 (YTHDF3) reader protein, thereby revealing a new role for YTHDF3 in shaping the selectivity of stress response. Our findings describe a previously unappreciated function for RNA m6A modification in the oxidative-stress response and expand the breadth of physiological roles of m6A.

molecular biology↗

bioRxiv2018

Absolute quantification of translational regulation and burden using combined sequencing approaches

Translation of mRNAs into proteins is a key cellular process. Ribosome binding sites and stop codons provide signals to initiate and terminate translation, while stable secondary mRNA structures can induce translational recoding events. Fluorescent proteins are commonly used to characterize such elements but require the modification of a parts natural context and allow only a few parameters to be monitored concurrently. Here, we develop an approach that combines ribosome profiling (Ribo-seq) with quantitative RNA sequencing (RNA-seq) to enable the high-throughput characterization of genetic parts controlling translation in absolute units. We simultaneously measure 743 translation initiation rates and 746 termination efficiencies across the Escherichia coli transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot structure. By analyzing the transcriptional and translational response, we discover that sequestered ribosomes at the pseudoknot contribute to a {sigma}32-mediated stress response, codon-specific pausing, and a drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches towards a comprehensive and quantitative understanding of gene regulation and burden in living cells.

synthetic biology↗