The template specificity of bacteriophage Phi6 RNA polymerase

Abstract

Bacteriophage Φ6 contains three double-stranded RNA (dsRNA) genomic segments, L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The virion RNA polymerase in the core particle transcribes segments M and S in vitro. Segment L is transcribed poorly because its transcript starts with GU instead of GG found on segments S and M. Transcription in vivo is modified by the binding of host protein YajQ to the outside the core particle so that segment L is transcribed well. This mechanism is the determinant of the temporal control of gene expression in Φ6. Mutants of Φ6 have been isolated that are independent of YajQ for transcription of segment L. The mutations are found in the gene of the viral polymerase or the major capsid protein or both. These mutants are capable of transcribing segment L with the GU start or GA or GC. The same is found to be true when YajQ is added to wild-type particles. Minus-strand synthesis has restrictions that are different from that of plus-strand synthesis, and YajQ or mutations to independence do not modify minus-strand synthesis behavior. Purified polymerase P2 is able to transcribe dsRNA, but transcription behavior of segment L by both wild-type and mutant polymerases is different from that seen in capsid structures. Adding YajQ to purified polymerase does not change its transcription specificity.

Fig 1

Transcriptase and minus-strand synthesis in wild-type particles packaging wild-type plus strands of segments S and M along with modified segment L plus strands that would result in transcripts starting with sequence GU, GA, GC, or GG. Incubation conditions are with or without the addition of purified protein YajQ. Bands labeled with lowercase letters are plus strands, while those labeled with uppercase letters contain dsRNA.

Fig 2

Transcriptase activity of wild-type (wt) and mutant nucleocapsids prepared from phage. Note that manganese ions activate L transcription but that YajQ-independent mutations in P2 are needed in normal magnesium buffers. The mutation K185R was found in several cases but was not essential for independence of YajQ.

Fig 3

Transcriptase and minus-strand synthesis in wild-type (produced by plasmid pLM687) and YajQ-independent mutant particles (produced by plasmid pLM3762) packaging wild-type plus strands of segments S and M along with modified segment L plus strands that would result in transcripts starting with sequence GU, GA, GC, or GG. The mutant particles contain P2 with mutation L612R.

Fig 4

Transcriptase and minus-strand synthesis in wild-type particles packaging wild-type plus strands of segments S and M along with modified segment L plus strands that would result in transcripts starting with sequence GU, GA, GC, or GG. Reactions were performed under normal conditions of 1 mM manganese and 5 mM magnesium ions or at a high (3 mM) manganese ion concentration with 1 mM magnesium ions.

Fig 5

Minus-strand synthesis in wild-type (pLM687) and YajQ-independent mutant (pLM3762) particles incubated with plus strands of S, M, and L terminated so as to produce minus strands beginning with GU or the normal AG. It is seen that minus-strand synthesis is normal and is not improved in the mutant particles. Plus-strand synthesis is as expected. The level of dsRNA labeling reflects the degree of minus-strand synthesis.

Fig 6

Minus- and plus-strand synthesis by core particles with mutant polymerase using S templates with altered 3′ termini resulting in varied 5′ sequences of the new minus strands. Plasmid pLM763 was cut with XbaI, HindIII, EcoRI, or SmaI with or without subsequent treatment with mung bean nuclease. All of the cuts treated with mung bean nuclease would have minus-strand transcripts with G as the second nucleotide. XbaI and EcoRI cuts without mung bean nuclease treatment would not have G as the second nucleotide. Segments M and L were wild type.

Fig 7

Transcription of normal dsRNA by purified wild-type (LM4801) and mutant (LM4820) polymerase at increasing protein concentrations.

Fig 8

Minus-strand synthesis by purified wild-type polymerase P2 on templates of S prepared from plasmid pLM763 as in Fig. 5.