Abstract
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 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 interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded Ecoli 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.
Highlights
In transcription initiation, the RNA polymerase (RNAP) holoenzyme binds promoter DNA by making sequence-specific interactions with core promoter elements and unwinds a turn of promoter DNA forming an RNAP-promoter open complex (RPo) containing a single-stranded “transcription bubble.” RNAP selects a transcription start site (TSS) by placing the startsite nucleotide and the nucleotide of the “template DNA strand” into the RNAP active-center product site (P site) and addition site (A site), respectively, and binding an initiating entity in the RNAP active-center P site (Fig. 1A)
The promoter-sequence determinants for primer-dependent initiation in E. coli, we modified a massively parallel reporter assay previously developed in our laboratory, termed massively systematic transcript end readout (MASTER) [12, 13], in order to detect both primer-independent and primer-dependent initiation, to differentiate between primer-independent and primer-dependent initiation, and to define primer lengths in primer-dependent initiation (Fig. 1B)
We treated RNAs in parallel with RNA 5′ pyrophosphohydrolase (Rpp) to detect RNAs generated by primer-independent initiation and with polynucleotide kinase (PNK), which converts a 5′-OH to a 5′-p, to detect RNAs generated by primer-dependent initiation (Fig. 1B)
Summary
The RNA polymerase (RNAP) holoenzyme binds promoter DNA by making sequence-specific interactions with core promoter elements and unwinds a turn of promoter DNA forming an RNAP-promoter open complex (RPo) containing a single-stranded “transcription bubble.” RNAP selects a transcription start site (TSS) by placing the startsite nucleotide and the nucleotide of the “template DNA strand” into the RNAP active-center product site (P site) and addition site (A site), respectively, and binding an initiating entity in the RNAP active-center P site (Fig. 1A). Crystal structures explain the structural basis of sequence dependence at the promoter position immediately upstream of the primer binding site, namely, interchain base stacking between the promoter template-strand nucleotide and primer 5′ nucleotide. The results demonstrate that most, if not all, primer-dependent initiation in E. coli involves use of a dinucleotide as the initiating entity and identify 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. We report crystal structures of transcription-initiation complexes containing dinucleotide primers that reveal the structural basis for a purine at the template-strand position immediately upstream of the primer binding site (RTSS−2): namely, more extensive, and likely more energetically favorable, base stacking between the template-strand base and the primer 5′ base
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