Interstellar bugs

Abstract

<h3>Abstract</h3> 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 <i>ab initio</i> mechanism for altering the RNA 5′ end. In <i>Escherichia coli</i> 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 <i>E. coli</i>. The results show primer-dependent initiation in <i>E. coli</i> 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, Y_TSS-2N_TSS-1N_TSSW_TSS+1, where TSS is the transcription start site, N_TSS-1N_TSS 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:R_TSS-2, where R is purine) exerts its effect through the base on the DNA template strand (R_TSS-2) through inter-chain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally-encoded <i>E</i>. <i>coli</i> 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.