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

Abstract

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 single-stranded DNA-binding protein (EcoSSB) is required for N4 early transcription in vivo, as well as for in vitro transcription on super-coiled 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.

Fig. 1.

Effect of EcoSSB on N4 vRNAP runoff transcription of the ssDNA template 12-P2–52. (A) Where indicated, EcoSSB and vRNAP were present at 1 μM and 10 nM, respectively. (B) A 100-μl complete transcription mixture containing 1 nM template but no EcoSSB was divided into four aliquots (I, II, III, and IV) before incubation at 37°C. I, no additions; II, 50 nM template was added after 20 min incubation; III, 1 μM EcoSSB was added before incubation; and IV, 1 μM EcoSSB was added after 20 min of incubation. At the indicated times, 5-μl aliquots were analyzed.

Fig. 2.

Effect of EcoSSB on the state of the DNA template and the RNA product. (A and B) Transcription reactions contained 5 nM unlabeled 12-P2–52 DNA template and [α-³²P]UTP. (C and D) Transcription reactions contained 5 nM 5′ ³²P-labeled template and unlabeled NTPs. Where indicated, EcoSSB was present at 1 μM. After a 15-min incubation, each mixture was divided into three aliquots and treated for 15 min at 37°C with no additions, RNase H, or Nuclease S1, as indicated, and analyzed by native PAGE.

Fig. 3.

(A and B) Effect of template length on EcoSBB activation. Runoff transcription reactions contained 1 nM template, 1 μM EcoSSB, and 1 μM mini-vRNAP and were incubated for 10 min at 37°C. (C) Interactions of the nascent transcript with mini-vRNAP and EcoSSB. (Upper) DNA template and nascent RNA arrangement in SEC. DNA templates bearing a single A in the transcribed region at the indicated positions numbered relative to the transcription start site (+1) were used in mini-vRNAP transcription reactions with 5-I-UTP, GTP, and [α-³²P]ATP. Arrowhead, position of the active center in the SEC. RNA is represented by a line. IU at positions –10 and –17 of the RNA in SEC is shown. (Lower) Crosslinking of the nascent RNA containing IU at different positions to mini-vRNAP and EcoSSB in SEC. Positions containing IU in nascent RNA of the SEC are numbered relative to the 3′ end (–1). Arrows indicate RNA crosslinked to mini-vRNAP and to EcoSSB. IU, 5-iodo-uridine.

Fig. 4.

Comparison of EcoSSB and Hsmt SSB. (A) Amino acid sequences of EcoSSB and Hsmt SSBs (7, 30). Conserved amino acids are shown in bold. The EcoSSB acidic C terminus is underlined. (B) Effect of Eco and Hsmt SSBs on vRNAP transcription. (C) DNase I footprinting of template DNA with EcoSSB and Hsmt SSB. Converging arrows indicate the promoter hairpin inverted repeat.

Fig. 5.

Mutant and chimeric SSB proteins and their effect on vRNAP transcription. (A) Schematic representation of mutant and chimeric proteins (12). DNA-binding domains (striped); EcoSSB C-terminal 10-aa region (filled); linker sequence between Eco SSB DNA-binding domain and the C-terminal 10-aa region (open); and Hsmt SSB unique N-terminal and C-terminal sequences (shaded). (B) The effect of mutant and chimeric SSB proteins on vRNAP transcription. Symbols are presented to the right of schematic representation in A. (C) Sequence of the C-terminal 13 aa of the EcoSSB (7), P1 SSB (13), and F SSB (33). (D) Effect of P1 SSB and F SSB on vRNAP transcription. Reaction mixtures containing no SSB or the indicated concentrations of different SSB proteins, 3 nM 12-P2–52 DNA template and 1 μM mini-vRNAP were incubated for 5 min at 37°C, and the products were analyzed on 8 M urea/8% polyacrylamide gels.