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Journal of Bacteriology, October 2004, p. 6864-6875, Vol. 186, No. 20
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.20.6864-6875.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Ribosomes and Translation Initiation Factors from the Gram-Positive Soil Bacterium Streptomyces lividans

J. Michael Day{dagger} and Gary R. Janssen*

Department of Microbiology, Miami University, Oxford, Ohio

Received 14 December 2003/ Accepted 22 July 2004

A primer extension inhibition (toeprint) assay was developed using ribosomes and ribosomal subunits from Streptomyces lividans. This assay allowed the study of ribosome binding to streptomycete leaderless and leadered mRNA. Purified 30S subunits were unable to form a ternary complex on aph leaderless mRNA, whereas 70S ribosomes could form ternary complexes on this mRNA. 30S subunits formed ternary complexes on leadered aph and malE mRNA. The translation initiation factors (IF1, IF2, and IF3) from S. lividans were isolated and included in toeprint and filter binding assays with leadered and leaderless mRNA. Generally, the IFs reduced the toeprint signal on leadered mRNA; however, incubation of IF1 and IF2 with 30S subunits that had been washed under high-salt conditions promoted the formation of a ternary complex on aph leaderless mRNA. Our data suggest that, as reported for Escherichia coli, initiation complexes with leaderless mRNAs might use a novel pathway involving 70S ribosomes or 30S subunits bound by IF1 and IF2 but not IF3. Some mRNA-ribosome-initiator tRNA reactions that yielded weak or no toeprint signals still formed complexes in filter binding assays, suggesting the occurrence of interactions that are not stable in the toeprint assay.

* Corresponding author. Mailing address: Department of Microbiology, Miami University, Oxford, OH 45056. Phone: (513) 529-1694. Fax: (513) 529-2431. E-mail: janssegr{at}muohio.edu.

{dagger} Present address: USDA, Agricultural Research Service, Southeast Poultry Research Laboratory, Athens, GA 30605.

Journal of Bacteriology, October 2004, p. 6864-6875, Vol. 186, No. 20
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.20.6864-6875.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Brock, J. E., Paz, R. L., Cottle, P., Janssen, G. R. (2007). Naturally Occurring Adenines within mRNA Coding Sequences Affect Ribosome Binding and Expression in Escherichia coli. J. Bacteriol. 189: 501-510 [Abstract] [Full Text]  


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