Structural basis for initiation of transcription from an RNA polymerase–promoter complex

Abstract

Although the single-polypeptide-chain RNA polymerase from bacteriophage T7 (T7RNAP), like other RNA polymerases, uses the same mechanism of polymerization as the DNA polymerases, it can also recognize a specific promoter sequence, initiate new RNA chains from a single nucleotide, abortively cycle the synthesis of short transcripts, be regulated by a transcription inhibitor, and terminate transcription. As T7RNAP is homologous to the Pol I family of DNA polymerases, the differences between the structure of T7RNAP complexed to substrates and that of the corresponding DNA polymerase complex provides a structural basis for understanding many of these functional differences. T7RNAP initiates RNA synthesis at promoter sequences that are conserved from positions -17 to +6 relative to the start site of transcription. The crystal structure at 2.4 A resolution of T7RNAP complexed with a 17-base-pair promoter shows that the four base pairs closest to the catalytic active site have melted to form a transcription bubble. The T7 promoter sequence is recognized by interactions in the major groove between an antiparallel beta-loop and bases. The amino-terminal domain is involved in promoter recognition and DNA melting. We have also used homology modelling of the priming and incoming nucleoside triphosphates from the T7 DNA-polymerase ternary complex structure to explain the specificity of T7RNAP for ribonucleotides, its ability to initiate from a single nucleotide, and the abortive cycling at the initiation of transcription.

Protein–DNA Electrostatics

“…The specific complex formed by the T7 RNA-polymerase and its target sequence (a gene promoter) has been studied by crystallography [53] [54]. The first interaction will include, typically, direct hydrogen bonds to base pairs and Van der Waals interactions [55,15].…”

Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase

“…Loading of DNA within the active site accompanies 'open complex' formation (3,5,6). The initial phase of transcription is inefficient and error-prone yielding short transcripts of 8 to 11nt in length, termed 'abortive initiation' (8)(9)(10)(11)(12)(13)(14). Further structural rearrangements result in promoter escape and finally RNAP enters the elongation phase (15).…”

T7 RNA polymerase transcription complex: What you see is not what you get

“…The specificity loop is a phage RNAP-specific feature, which in the open promoter complex recognizes the doublestranded DNA 10-12 base pairs upstream of the transcription initiation start point. Strikingly, 2 amino acids within the 7-aa fragment that harbors the crosslink site are known to contact the promoter at positions Ϫ7, Ϫ10, and Ϫ11 and are critical in specific promoter recognition (16,17). Thus, it seems that during promoter clearance, the contacts between the specificity loop and the upstream-promoter DNA are broken, and new contacts with RNA and possibly DNA at the upstream edge of the transcription bubble are established.…”

“…16). The proposed trajectory results in few clashes, is consistent with much of the biochemical and genetic data, and is not consistent with the RNA-exit pathway suggested by the structure of the T7 RNAP initiation complex (12).…”

“…This critical position, where RNA is displaced from the hybrid, seems to contribute to elongation complex stability. By using a panel of chemical-mapping techniques in combination with RNAP mutants, the crosslink site was localized to within 7 T7 RNAP amino acids in the so-called specificity loop (16). The specificity loop is a phage RNAP-specific feature, which in the open promoter complex recognizes the doublestranded DNA 10-12 base pairs upstream of the transcription initiation start point.…”