S1 is the largest ribosomal protein, and is vitally important for the cell. S1 is also a subunit of Qb replicase, the RNA-directed RNA polymerase of bacteriophage Qb. In both protein and RNA syntheses, S1 is commonly believed to bind to a template RNA at the initiation step, and not to be involved in later events. Here, we show that in Qb replicase-mediated RNA synthesis, S1 functions at the termination step by promoting release of the product strand in a single-stranded form. This function is fulfilled by the N-terminal fragment comprising the first two S1 domains. The results suggest that S1 might also have a role other than mRNA binding in the ribosome.

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

ABSTARCT The high resolution crystal structure of bacterial ribosome was determined more than 10 years ago; however, it contains no information on the structure of the largest ribosomal protein, S1. This unusual protein comprises six flexibly linked domains; therefore, it lacks a fixed structure and this prevents the formation of crystals. Besides being a component of the ribosome, protein S1 also serves as one of the four subunits of Qβ replicase, the RNA-directed RNA polymerase of bacteriophage Qβ. In each case, the role of this RNA-binding protein has been thought to consist in holding the template close to the active site of the enzyme. In recent years, a breakthrough was made in studies of protein S1 within Qβ replicase. This includes the discovery of its paradoxical ability to displace RNA from the replicase complex and determining the crystal structure of its fragment capable of performing this function. The new findings call for a re-examination of the contribution of protein S1 to the structure and function of the ribosome.

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent RNA polymerase (β-subunit) and the host-encoded translation elongation factors, EF-Ts and EF-Tu as well as the ribosomal protein S1. The Qβ replicase has an extraordinary ability to exponentially amplify RNA in vivo and in vitro. A prerequisite for this is release of product and template RNA as single strands that can serve as new templates in subsequent rounds of replication. The role of S1 in the Qβ replicase is not clear. Recently, S1 was found to promote release of single-stranded product in Qβ replicase-mediated RNA synthesis. We have undertaken NMR spectroscopy and crystallization trials to improve our understanding of distinct S1 domains in solution as well as the ribosomeand replicase-binding properties of S1. Expression of distinct S1 domains for NMR spectroscopy has been optimized by use of autoinduction and results in high yields of [13C15N]-labelled protein fragments. These have proven very suitable for NMR studies and spectra revealed both ordered and disordered regions in the protein. Studies are ongoing. The structure of the Qβ core complex was recently determined at 2.5Å resolution. Thus, co-crystallization of the Qβ core in complex with S1 domains was undertaken and different crystal forms were obtained. These initial crystals diffracted to 3.2Å resolution and data processing as well as further optimization of the crystals is ongoing. S1 is thought to bind the β-subunit close to a region lined with basic amino acids, which potentially could facilitate interactions with the template RNA backbone and split it from the product strand. We demonstrate that neutralization of these basic amino acids indeed decrease or abolish infectivity of the Qβ phage. However, only one mutation, R503A affects the exponential replication in vitro. Crystallization of the Qβ holo enzyme bound to a truncated legitimate RNA template will be the next step for investigation of the mechanism of exponential RNA amplification by Qβ replicase.