Biology subjects

Skalenko, K. S.

Publications and source records attributed to Skalenko, K. S..

3 recordsLinked to original sources

bioRxiv2023

Linking genotypic and phenotypic changes in the LTEE using metabolomics

1Changes in an organisms environment, genome, or gene expression patterns can lead to changes in its metabolism. The metabolic phenotype can be under selection and contributes to adaptation. However, the networked and convoluted nature of an organisms metabolism makes relating mutations, metabolic changes, and effects on fitness challenging. To overcome this challenge, we use the Long-Term Evolution Experiment (LTEE) with E. coli as a model to understand how mutations can eventually affect metabolism and perhaps fitness. We used mass-spectrometry to broadly survey the metabolomes of the ancestral strains and all 12 evolved lines. We combined this metabolic data with mutation and expression data to suggest how mutations that alter specific reaction pathways, such as the biosynthesis of nicotinamide adenine dinucleotide, might increase fitness in the system. Our work provides a better understanding of how mutations might affect fitness through the metabolic changes in the LTEE and thus provides a major step in developing a complete genotype-phenotype map for this experimental system.

evolutionary biology↗

bioRxiv2022

Structural and mechanistic basis of σ-dependent transcriptional pausing

In {sigma}-dependent transcriptional pausing, the transcription initiation factor {sigma}, translocating with RNA polymerase (RNAP), makes sequence-specific protein-DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in {sigma}-dependent pausing, the RNAP active center can access off-pathway "backtracked" states that are substrates for the transcript-cleavage factors of the Gre family, and on-pathway "scrunched" states that mediate pause escape. Here, using site-specific protein-DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of {sigma} relative to DNA at the{lambda} PR promoter, we show directly that {sigma}-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2-4 bp, and that {sigma}-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2-3 bp. Analogous experiments with a library of 47 ([~]16,000) transcribed-region sequences show that the state scrunched by 2-3 bp--and only that state--is associated with the consensus sequence, T-3N-2Y-1G+1, (where -1 corresponds to the position of the RNA 3 end), which is identical to the consensus for pausing in initial transcription, and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T-3 resides in the DNA nontemplate strand. A cryo-EM structure of a complex engaged in {sigma}-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T-3 of the consensus sequence exerts its effects by facilitating scrunching.

biochemistry↗

bioRxiv2021

Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

Chemical modifications of RNA 5' ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates (NTPs) for use as initiating entities, providing an ab initio mechanism for altering the RNA 5' end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here we use massively systematic transcript end readout ("MASTER") to detect and quantify RNA 5' ends generated by primer-dependent initiation for ~410 (~1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through inter-chain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally-encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position, but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.

biochemistry↗