A primary transcript in spinach chloroplasts that completely lacks a 5′ untranslated leader region

Bennett, Dorothy C.; Rogers, Sharon A.; Chen, Liang-Jwu; Orozco, Emil M.

doi:10.1007/bf00017728

Cited by 16 publications

(8 citation statements)

References 32 publications

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“…Spinach atpB mRNA has five 5' termini, four of which result from alternative initiation sites. Interestingly, one of these termini maps precisely at the start of the coding region, and yet this RNA copurified with crude polysomal fractions (2). In at least one case in higher plants, the location of the 5' terminus can affect the translatability of the message.…”

Section: Resultsmentioning

confidence: 99%

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Sakamoto¹,

Sturm²,

Kindle³

et al. 1994

Mol. Cell. Biol.

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Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6lf complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription ofpetD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidAl fusion genes into the chloroplast genome (uid4 is an Escherichia coli gene that encodes ,-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uid4 transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA4 transcript accumulated but no monocistronic uid4 transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. ,1-Glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uid4 transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.Higher-plant chloroplast genes are organized as operons that are transcribed into polycistronic primary transcripts. Splicing and other endo-and exonucleolytic cleavages subsequently occur to generate mature transcripts (1,9,12,32). One such operon in higher plants contains the petD gene, which encodes subunit IV of the cytochrome bdlf complex. Its genomic location is conserved in higher plants (13,30) and cyanobacteria (15); however, its mode of expression varies. In spinach chloroplasts, a primary transcript containing the psbB, psbH, petB, and petD coding regions is processed into 18 RNA species, including monocistronicpsbB andpsbH transcripts and a dicistronic petB-petD transcript (32). As no monocistronic petD mRNA has been detected in spinach chloroplasts, the coding region is presumed to be translated from the dicistronic message. The transcription ofpetD RNA in maize chloroplasts also occurs as part of the psbB operon, but the major translatable form of the petD message is monocistronic. Nonetheless, polycistronic transcripts can be immunoprecipitated from maize polysomes with subunit IV-spe...

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Section: Resultsmentioning

confidence: 99%

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

Sakamoto¹,

Sturm²,

Kindle³

et al. 1994

Mol. Cell. Biol.

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“…Translation of the tobacco atpE cistron may also require two or more cis -elements. In spinach chloroplasts, the atpB / E transcript lacking its 5′-UTR was found in crude polysomal fractions (48). Our study has clearly indicated that removal of the atpB 5′-UTR completely arrests translation of the atpB cistron but not the atpE cistron (see Figure 3, lanes ‘–‘).…”

Section: Discussionmentioning

confidence: 99%

The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

Suzuki

Kuroda

Yukawa

et al. 2011

Nucleic Acids Research

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The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA, and that the efficiency of atpB translation is higher than that of atpE translation. When the atpB 5′-UTR was replaced with lower efficiency 5′-UTRs, atpE translation was higher than atpB translation. Removal of the entire atpB 5′-UTR arrested atpB translation but atpE translation still proceeded. Introduction of a premature stop codon in the atpB cistron did not abolish atpE translation. These results indicate that atpE translation is independent of atpB translation. Mutation analysis showed that the atpE cistron possesses its own cis-element(s) for translation, located ~25 nt upstream from the start codon.

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“…Although this is unconventional because such Es-655 cherichia coli-like promoters occur at -10 from the transcription start site, it is possible that 5 bases are removed from the psbH primary transcripts. However, recent transcription experiments with the Chlamydomonas atpB gene show initiation from a promoter which does not fit the conventional E. coli promoter consensus sequence [25 In contrast, the atpB gene of vascular plants il tiates transcription from a consensus E. col#like ,ranscriptional promoter [6,9,12]. Many othc: Chlamydomonas chloroplast genes, whose transcriptional start sites have been identified, were also found to lack conventional E. col#like promoters [25].…”

Section: Discussionmentioning

confidence: 99%

The psbB gene cluster of the Chlamydomonas reinhardtii chloroplast: sequence and transcriptional analyses of psbN and psbH

Johnson

Schmidt

1993

Plant Mol Biol

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We have sequenced and characterized the complete psbB gene cluster of Chlamydomonas reinhardtii chloroplast DNA. Although the petB and petD genes are located elsewhere, the sequential order of psbB, ORF31, psbN and psbH is identical to that of the psbB operon in higher plants. Also, intergenic non-coding regions are much larger in the Chlamydomonas gene cluster. Northern blot analyses indicate the formation of dicistronic transcripts of psbB and ORF31 and monocistronic transcripts of psbN and psbH. It is unclear whether a psbB operon is transcribed to yield a large polycistronic precursor but northern blot analysis with total RNA from cells grown at 15 degrees C does not detect an increased complexity of the transcripts, as has been found in studies of the psbB operon of higher plants. From primer extension and nuclease protection assays, it is apparent that 5' and 3' processing of the primary psbH transcript results in the accumulation of a heterogenous population of mRNAs. Northern blot analyses reveal transcription of Chlamydomonas psbN and show that its mRNA is much larger than that identified in liverwort and pea. The sequence identities of the PSII-H and PSII-N polypeptides as compared to their vascular plant counterparts is 50 to 62%. While the amino acid sequences of PSII-H and PSII-N proteins are significantly conserved, the mass of PSII-H from Chlamydomonas is significantly larger.

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A primary transcript in spinach chloroplasts that completely lacks a 5′ untranslated leader region

Cited by 16 publications

References 32 publications

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing.

The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

The psbB gene cluster of the Chlamydomonas reinhardtii chloroplast: sequence and transcriptional analyses of psbN and psbH

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