E. coli SSB Activates N4 Virion RNA Polymerase Promoters by Stabilizing a DNA Hairpin Required for Promoter Recognition

432

556

Nucleic acid hairpins provide a powerful model system for probing the formation of secondary structure. We report a systematic study of the kinetics and thermodynamics of the folding transition for individual DNA hairpins of varying stem length, loop length, and stem GC content. Folding was induced mechanically in a highresolution optical trap using a unique force clamp arrangement with fast response times. We measured 20 different hairpin sequences with quasi-random stem sequences that were 6 -30 bp long, polythymidine loops that were 3-30 nt long, and stem GC content that ranged from 0% to 100%. For all hairpins studied, folding and unfolding were characterized by a single transition. From the force dependence of these rates, we determined the position and height of the energy barrier, finding that the transition state for duplex formation involves the formation of 1-2 bp next to the loop. By measuring unfolding energies spanning one order of magnitude, transition rates covering six orders of magnitude, and hairpin opening distances with subnanometer precision, our results define the essential features of the energy landscape for folding. We find quantitative agreement over the entire range of measurements with a hybrid landscape model that combines thermodynamic nearest-neighbor free energies and nanomechanical DNA stretching energies.DNA hairpin ͉ energy landscape ͉ force clamp ͉ optical tweezers ͉ single molecule H airpins formed from self-complementary sequences supply a model system for studying folding and duplex formation, the most fundamental processes for generating structure in nucleic acids. Using hairpins, repeated measurements can be made on the same molecule, facilitating single-molecule studies. Furthermore, by simply altering the nucleotide sequence, physical properties such as folding energies, kinetic rates, and distances to transition states all can be changed systematically. Hairpins also play essential roles in vivo. DNA hairpins bind proteins to regulate transcription (1), and hairpin intermediates are involved in both replication and recombination (2, 3). RNA hairpins form tertiary contacts (4), bind to proteins (2), regulate transcription (5), and mediate RNA interference (6). Understanding the factors that influence hairpin folding should therefore not only elucidate principles of structure formation in nucleic acids but may also shed light on the biological roles played by these structures.Extensive calorimetric and melting studies have been carried out to generate predictive rules for the thermodynamic stability of arbitrary nucleic acid duplexes (7). The kinetic properties of duplex formation, however, remain less well understood, particularly those related to the nature of the transition state. Temperature-jump studies of annealing in short duplexes have been interpreted in terms of the nucleation of a transition state consisting of Ϸ1-3 bp, followed by zippering of the remaining stem (8). This interpretation, however, rests on the assumption that the enthalpy of activation arises ...

“…For hairpins with random stem sequences, the barrier height is set mostly by the energy for unfolding or reforming the loop under load. Expressing the lifetime as 1 …”

Section: Resultsmentioning

confidence: 99%

“…Hairpins also play essential roles in vivo. DNA hairpins bind proteins to regulate transcription (1), and hairpin intermediates are involved in both replication and recombination (2,3). RNA hairpins form tertiary contacts (4), bind to proteins (2), regulate transcription (5), and mediate RNA interference (6).…”

mentioning

confidence: 99%

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

Woodside

Behnke-Parks²,

Larizadeh³

et al. 2006

432

556

“…However, although T7 RNAP recognizes its promoters in double-stranded templates (5), vRNAP transcribes promotercontaining, single-stranded templates with in vivo specificity (6), recognizing a 3-base loop-5-bp stem hairpin structure and specific sequences at the promoter (7). In vivo, vRNAP promoter recognition requires template supercoiling (8) to drive extrusion of the promoter hairpins from the double-stranded N4 genome (9,10) and Escherichia coli single-stranded DNA-binding protein to stabilize single-stranded regions surrounding the site of transcription initiation (11). We have used single-stranded templates to identify unambiguously the site of transcription initiation as well as the sequence and structural DNA determinants of vRNAP-promoter interaction.…”

mentioning

confidence: 99%

Bacteriophage N4 virion RNA polymerase interaction with its promoter DNA hairpin

Davydova

Santangelo²,

Rothman-Denes³

2007

Bacteriophage N4 minivirion RNA polymerase (mini-vRNAP), the RNA polymerase (RNAP) domain of vRNAP, is a member of the T7-like RNAP family. Mini-vRNAP recognizes promoters that comprise conserved sequences and a 3-base loop-5-base pair (bp) stem DNA hairpin structure on single-stranded templates. Here, we defined the DNA structural and sequence requirements for minivRNAP promoter recognition. Mini-vRNAP binds a 20-nucleotide (nt) N4 P2 promoter deoxyoligonucleotide with high affinity (K d ‫؍‬ 2 nM) to form a salt-resistant complex. We show that mini-vRNAP interacts specifically with the central base of the hairpin loop (؊11G) and a base at the stem (؊8G) and that the guanine 6-keto and 7-imino groups at both positions are essential for binding and complex salt resistance. The major determinant (؊11G), which must be presented to mini-vRNAP in the context of a hairpin loop, appears to interact with mini-vRNAP Trp-129. This interaction requires template single-strandedness at positions ؊2 and ؊1. Contacts with the promoter are disrupted when the RNA product becomes 11-12 nt long. This detailed description of vRNAP interaction with its promoter hairpin provides insights into RNAPpromoter interactions and explains how the injected vRNAP, which is present in one or two copies, recognizes its promoters on a single copy of the injected genome.T7-like RNA polymerase B acteriophage N4 virion RNA polymerase (vRNAP), which is responsible for the transcription of the phage early genes, is present in virions in one or two copies and is injected into the host together with the phage genome at the onset of infection (1, 2). vRNAP is a 3,500-aa polypeptide that lacks extensive sequence similarity to either of the two known families of DNA-dependent RNAPs (3). Using controlled trypsin proteolysis and catalytic autolabeling, we defined a stable and transcriptionally active 1,106-aa domain (mini-vRNAP) located at the center of the vR-NAP polypeptide (3). Mini-vRNAP possesses the same initiation, elongation, termination, and product displacement properties as full-length vRNAP (3, 4).Mutational, biochemical, and phylogenetic analyses indicated that mini-vRNAP is a highly diverged member of the T7 RNAP family (3). However, although T7 RNAP recognizes its promoters in double-stranded templates (5), vRNAP transcribes promotercontaining, single-stranded templates with in vivo specificity (6), recognizing a 3-base loop-5-bp stem hairpin structure and specific sequences at the promoter (7). In vivo, vRNAP promoter recognition requires template supercoiling (8) to drive extrusion of the promoter hairpins from the double-stranded N4 genome (9, 10) and Escherichia coli single-stranded DNA-binding protein to stabilize single-stranded regions surrounding the site of transcription initiation (11). We have used single-stranded templates to identify unambiguously the site of transcription initiation as well as the sequence and structural DNA determinants of vRNAP-promoter interaction. The results reveal two major determinants of vRNAP hairpin rec...

“…Other SSBs (T7 gp 2.5, T4 gp 32, and N4 SSB) fail to stimulate vRNAP transcription in vitro because they destabilize the promoter hairpin (6). vRNAP transcripts generated from ssDNA templates are retained as RNA⅐DNA hybrids, suggesting that vRNAP cannot displace the RNA product (2).…”

mentioning

confidence: 99%

Escherichia coli single-stranded DNA-binding protein mediates template recycling during transcription by bacteriophage N4 virion RNA polymerase

Davydova

Rothman-Denes

2003

Coliphage N4 virion RNA polymerase (vRNAP), the most distantly related member of the T7-like family of RNA polymerases, is responsible for transcription of the early genes of the linear double-stranded DNA phage genome. Escherichia coli singlestranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on supercoiled DNA templates containing vRNAP promoters. In contrast to other DNA-dependent RNA polymerases, vRNAP initiates transcription on single-stranded, promoter-containing templates with in vivo specificity; however, the RNA product is not displaced, thus limiting template usage to one round. We show that EcoSSB activates vRNAP transcription at limiting single-stranded template concentrations through template recycling. EcoSSB binds to the template and to the nascent transcript and prevents the formation of a transcriptionally inert RNA:DNA hybrid. Using C-terminally truncated EcoSSB mutant proteins, human mitochondrial SSB (Hsmt SSB), phage P1 SSB, and F episome-encoded SSB, as well as a Hsmt-EcoSSB chimera, we have mapped a determinant of template recycling to the C-terminal amino acids of EcoSSB. T7 RNAP contains an amino-terminal domain responsible for binding the RNA product as it exits from the enzyme. No sequence similarity to this domain exists in vRNAP. Hereby, we propose a unique role for EcoSSB: It functionally substitutes in N4 vRNAP for the N-terminal domain of T7 RNAP responsible for RNA binding.B acteriophage N4 virion RNA polymerase (vRNAP), responsible for transcription of the phage early genes, is injected into the host cell with the phage genome upon infection (1). vRNAP is inactive on linear double-stranded templates but transcribes denatured genomic N4 DNA or promoter-containing single-stranded DNA (ssDNA) with in vivo specificity (2, 3). vRNAP promoters contain a 5-to 7-bp stem, 3-nt loop hairpin, and conserved sequences (3, 4).In vivo, N4 early transcription requires Escherichia coli DNA gyrase and E. coli ssDNA-binding protein (EcoSSB), which provide an ''active'' promoter structure (5). Other SSBs (T7 gp 2.5, T4 gp 32, and N4 SSB) fail to stimulate vRNAP transcription in vitro because they destabilize the promoter hairpin (6). vRNAP transcripts generated from ssDNA templates are retained as RNA⅐DNA hybrids, suggesting that vRNAP cannot displace the RNA product (2). We show that EcoSSB activates vRNAP transcription on ssDNA templates through displacement of the RNA product and, therefore, through template recycling.The 177-aa EcoSSB polypeptide consists of a 115-aa Nterminal domain responsible for DNA binding and an Ϸ50-aa Pro-and Gly-rich sequence followed by a 10-aa acidic tail, highly conserved in eubacterial SSBs (7,8). Human mitochondrial (Hsmt) SSB is similar in sequence and structure to the Nterminal domain of EcoSSB but lacks the conserved C-terminal sequence (9-11). We show that Hsmt SSB did not activate vRNAP transcription although it did not disrupt the promoter hairpin. We determined that the 10 C-terminal ami...