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J. Biol. Chem., Vol. 281, Issue 50, 38314-38321, December 15, 2006

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Translation Initiation of Cyanobacterial rbcS mRNAs Requires the 38-kDa Ribosomal Protein S1 but Not the Shine-Dalgarno Sequence

DEVELOPMENT OF A CYANOBACTERIAL IN VITRO TRANSLATION SYSTEM^*

Michinori Mutsuda¹ and Masahiro Sugiura²

From the Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan and the Graduate School of Natural Sciences, Nagoya City University, Nagoya 467-8501, Japan

Little is known about the biochemical mechanism of translation in cyanobacteria though substantial studies have been made on photosynthesis, nitrogen fixation, circadian rhythm, and genome structure. To analyze the mechanism of cyanobacterial translation, we have developed an in vitro translation system from Synechococcus cells using a psbAI-lacZ fusion mRNA as a model template. This in vitro system supports accurate translation from the authentic initiation site of a variety of Synechococcus mRNAs. In Synechococcus cells, rbcL and rbcS encoding the large and small subunits, respectively, of ribulose-1,5-bisphosphate carboxylase/oxygenase are co-transcribed as a dicistronic mRNA, and the downstream rbcS mRNA possesses two possible initiation codons separated by three nucleotides. Using this in vitro system and mutated mRNAs, we demonstrated that translation starts exclusively from the upstream AUG codon. Although there are Shine-Dalgarno-like sequences in positions similar to those of the functional Shine-Dalgarno elements in Escherichia coli, mutation analysis indicated that these sequences are not required for translation. Assays with deletions within the 5'-untranslated region showed that a pyrimidine-rich sequence in the –46 to –15 region is necessary for efficient translation. Synechococcus cells contain two ribosomal protein S1 homologues of 38 and 33 kDa in size. UV cross-linking and immunoprecipitation experiments suggested that the 38-kDa S1 is involved in efficient translation via associating with the pyrimidine-rich sequence. The present in vitro translation system will be a powerful tool to analyze the basic mechanism of translation in cyanobacteria.

Received for publication, May 15, 2006 , and in revised form, October 13, 2006.

^* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Present address: Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.

² To whom correspondence should be addressed: Sugiyama Human Research Center, Sugiyama Jogakuen University, Nagoya 464-8662, Japan. Tel.: 81-52781-7137; Fax: 81-52781-7196; E-mail: sugiura{at}nsc.nagoya-cu.ac.jp.

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