The petD gene is transcribed by functionally redundant promoters in Chlamydomonas reinhardtii chloroplasts

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

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Transcriptional Activity of the Telomeric Retrotransposon HeT-A in Drosophila melanogaster Is Stimulated as a Consequence of Subterminal Deficiencies at Homologous and Nonhomologous Telomeres

Molecular and Cellular Biology ◽

10.1128/mcb.00515-07 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4991-5001 ◽

Cited By ~ 7

Author(s):

Radmila Capkova Frydrychova ◽

Harald Biessmann ◽

Alexander Y. Konev ◽

Mikhail D. Golubovsky ◽

Jessica Johnson ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Promoter Activity ◽

Transcriptional Activity ◽

Position Effect ◽

Wild Type ◽

Global Effect ◽

Transcript Levels ◽

Telomere Position Effect

ABSTRACT Drosophila melanogaster telomeres have two DNA domains: a terminal array of retrotransposons and a subterminal repetitive telomere-associated sequence (TAS), a source of telomere position effect (TPE). We reported previously that deletion of the 2L TAS array leads to dominant suppression of TPE by stimulating in trans expression of a telomeric transgene. Here, we compared the transcript activities of a w transgene inserted between the retrotransposon and TAS arrays at the 2L telomere in genotypes with different lengths of the 2L TAS. In contrast to individuals bearing a wild-type 2L homologue, flies with a TAS deficiency showed a significant increase in the level of telomeric w transcript during development, especially in pupae. Moreover, we identified a read-through w transcript initiated from a retrotransposon promoter in the terminal array. Read-through transcript levels also significantly increased with the presence of a 2L TAS deficiency in trans, indicating a stimulating force of the TAS deficiency on retrotransposon promoter activity. The read-through transcript contributes to total w transcript, although most w transcript originates at the w promoter. While silencing of transgenes in nonhomologous telomeres is suppressed by 2L TAS deficiencies, suggesting a global effect, the overall level of HeT-A transcripts is not increased under similar conditions.

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3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2277-2285.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2277-2285

Author(s):

D B Stern ◽

K L Kindle

Keyword(s):

Chlamydomonas Reinhardtii ◽

Higher Plants ◽

Transcription Termination ◽

Protein Coding ◽

Coding Regions ◽

In Vitro Processing ◽

Mrna Precursor ◽

Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

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Characterization of transposable element-associated mutations that alter yeast alcohol dehydrogenase II expression

Molecular and Cellular Biology ◽

10.1128/mcb.3.1.20-31.1983 ◽

1983 ◽

Vol 3 (1) ◽

pp. 20-31

Author(s):

V M Williamson ◽

D Cox ◽

E T Young ◽

D W Russell ◽

M Smith

Keyword(s):

Alcohol Dehydrogenase ◽

Dna Sequences ◽

Target Sequence ◽

Direct Repeats ◽

Wild Type ◽

Coding Region ◽

Base Pairs ◽

Yeast Saccharomyces Cerevisiae ◽

Delta Sequence ◽

Flanking Sequences

Seven cis-dominant, constitutively expressed mutations of the normally glucose-repressible isozyme of alcohol dehydrogenase (ADHII) from the yeast Saccharomyces cerevisiae are caused by insertion of transposable elements from the Ty1 family in front of the ADHII structural gene (ADR2) (V. M. Williamson, E. T. Young, and M. Ciriacy, Cell 23:605-614, 1981). We cloned ADR2 with its associated Ty1 element from five S. cerevisiae strains carrying these mutations. Comparison of the Ty1 elements by heteroduplex studies and restriction enzyme analyses indicated that four were very similar; the fifth, although the same size as the others (about 5.6 kilobases), differed by the presence of two large substitutions of approximately 1 and 2 kilobases. The DNA sequences of the terminal direct repeats (deltas) were very homologous but not identical and were similar to previously reported Ty1 element direct repeats. We determined the 5'-flanking sequences of the ADR2 gene isolated from a wild-type strain and from five Ty1-associated mutations. The 5-base pair target sequence at the site of Ty1 insertion was present at both ends of each Ty1 element. The sites of insertion of the elements were all different and occurred from 125 to 210 base pairs in front of the coding region of ADR2. The 5' end of the major transcript as determined by S1 mapping was the same in wild-type cells and in Ty1-associated constitutive mutants and was approximately 54 base pairs upstream from the coding region. ADR2 transcripts were not detected when a solo delta sequence was present in the 5'-flanking region of this gene.

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Modulation of transcriptional activity and stable complex formation by 5'-flanking regions of mouse tRNAHis genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.105 ◽

1986 ◽

Vol 6 (1) ◽

pp. 105-115 ◽

Cited By ~ 21

Author(s):

M J Morry ◽

J D Harding

Keyword(s):

Transcriptional Activity ◽

Trna Gene ◽

Stable Complex ◽

Wild Type ◽

Flanking Sequence ◽

Coding Regions ◽

S 100 ◽

Flanking Region ◽

Flanking Regions

We determined the nucleotide sequences of three mouse tRNAHis genes and a tRNAGly gene present in two different lambda clones. One lambda clone contained two tRNAHis genes 600 base pairs (bp) apart in opposite orientations. The other clone contained a tRNAHis and a tRNAGly gene 569 bp apart in the same orientation. The coding regions of the three tRNAHis genes were identical to sequenced mammalian tRNAHis if posttranscriptional modifications are not considered. Notably, the three tRNAHis genes and a fourth gene previously sequenced by us contained within the flanking regions, various amounts of short, conserved 5' leader sequences and 3' trailer sequences directly abutting the coding regions. Otherwise the flanking regions were not homologous. Deletion mutants of one of the tRNAHis genes were constructed which contained 228, 99, 9, and 3 bp of the wild-type 5'-flanking region, respectively. Deletion of 5'-flanking sequences from positions -9 to -4 reduced transcriptional activity substantially (ca. fivefold) in a HeLa cell S-100 lysate. This effect was independent of the vector sequences in the deletion clone, implying that the region from -4 to -9 of the intact gene contains a positive modulatory element for transcription in vitro. The deletion mutant containing 3 bp of wild-type 5'-flanking sequence also had a greatly reduced ability to inhibit the transcription of a second tRNA gene in a competition assay. Thus, the normal 5'-flanking region influences the ability of the gene to form stable complexes with transcription factors. These data further indicate that a mammalian transcription extract is sensitive to 5'-flanking-region effects if a suitable tRNA gene is assayed.

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TCA1, a Single Nuclear-Encoded Translational Activator Specific for petA mRNA in Chlamydomonas reinhardtii Chloroplast

Genetics ◽

10.1093/genetics/159.1.119 ◽

2001 ◽

Vol 159 (1) ◽

pp. 119-132

Author(s):

K Wostrikoff ◽

Y Choquet ◽

F-A Wollman ◽

J Girard-Bascou

Keyword(s):

Chlamydomonas Reinhardtii ◽

Nuclear Gene ◽

Photosynthetic Electron Transport ◽

Cytochrome F ◽

Coding Region ◽

Reading Frame ◽

Transport Chain ◽

Nuclear Loci ◽

Translational Activator ◽

B6f Complex

Abstract We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5′ untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of cytochrome b6f complex petA mRNA. The high number of TCA1 alleles, together with the absence of genetic evidence for other nuclear loci controlling translation of the chloroplast petA gene, strongly suggests that TCA1 is the only trans-acting factor. We studied the assembly-dependent regulation of cytochrome f translation—known as the CES process—in TCA1-mutated contexts. In the presence of a leaky tca1 allele, we observed that the regulation of cytochrome f translation was now exerted within the limits of the restricted translational activation conferred by the altered version of TCA1 as predicted if TCA1 was the ternary effector involved in the CES process.

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3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2277 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2277-2285 ◽

Cited By ~ 50

Author(s):

D B Stern ◽

K L Kindle

Keyword(s):

Chlamydomonas Reinhardtii ◽

Higher Plants ◽

Transcription Termination ◽

Protein Coding ◽

Coding Regions ◽

In Vitro Processing ◽

Mrna Precursor ◽

Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

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TATA-Binding Protein–TATA Interaction Is a Key Determinant of Differential Transcription of Silkworm Constitutive and Silk Gland-Specific tRNAAla Genes

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1329-1343.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1329-1343 ◽

Cited By ~ 21

Author(s):

Ching Ouyang ◽

M. Juanita Martinez ◽

Lisa S. Young ◽

Karen U. Sprague

Keyword(s):

Promoter Activity ◽

Transcriptional Activity ◽

Binding Protein ◽

Tata Binding Protein ◽

Silk Gland ◽

Wild Type ◽

Heparin Resistance ◽

Transcription Complexes ◽

Differential Transcription

ABSTRACT We have investigated the contribution of specific TATA-binding protein (TBP)–TATA interactions to the promoter activity of a constitutively expressed silkworm tRNAC Ala gene and have also asked whether the lack of similar interactions accounts for the low promoter activity of a silk gland-specific tRNASG Ala gene. We compared TBP binding, TFIIIB-promoter complex stability (measured by heparin resistance), and in vitro transcriptional activity in a series of mutant tRNAC Ala promoters and found that specific TBP-TATA contacts are important for TFIIIB-promoter interaction and for transcriptional activity. Although the wild-type tRNAC Ala promoter contains two functional TBP binding sequences that overlap, the tRNASG Ala promoter lacks any TBP binding site in the corresponding region. This feature appears to account for the inefficiency of the tRNASG Alapromoter since provision of either of the wild-type TATA sequences derived from the tRNAC Ala promoter confers robust transcriptional activity. Transcriptional impairment of the wild-type tRNASG Ala gene is not due to reduced incorporation of TBP into transcription complexes since both the tRNAC Ala and tRNASG Ala promoters form transcription complexes that contain the same amount of TBP. Thus, the deleterious consequences of the lack of appropriate TBP-TATA contacts in the tRNASG Ala promoter must come from failure to incorporate some other essential transcription factor(s) or to stabilize the complete complex in an active conformation.

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Regulation of dev, an Operon That Includes Genes Essential for Myxococcus xanthus Development and CRISPR-Associated Genes and Repeats

Journal of Bacteriology ◽

10.1128/jb.00187-07 ◽

2007 ◽

Vol 189 (10) ◽

pp. 3738-3750 ◽

Cited By ~ 75

Author(s):

Poorna Viswanathan ◽

Kimberly Murphy ◽

Bryan Julien ◽

Anthony G. Garza ◽

Lee Kroos

Keyword(s):

Escherichia Coli ◽

Promoter Activity ◽

Myxococcus Xanthus ◽

Regulatory Elements ◽

Fruiting Bodies ◽

Wild Type ◽

Coding Region ◽

Upstream Gene ◽

Negative Element ◽

Growth Induction

ABSTRACT Expression of dev genes is important for triggering spore differentiation inside Myxococcus xanthus fruiting bodies. DNA sequence analysis suggested that dev and cas (CRISPR-associated) genes are cotranscribed at the dev locus, which is adjacent to CRISPR (clustered regularly interspaced short palindromic repeats). Analysis of RNA from developing M. xanthus confirmed that dev and cas genes are cotranscribed with a short upstream gene and at least two repeats of the downstream CRISPR, forming the dev operon. The operon is subject to strong, negative autoregulation during development by DevS. The dev promoter was identified. Its −35 and −10 regions resemble those recognized by M. xanthus σA RNA polymerase, the homolog of Escherichia coli σ70, but the spacer may be too long (20 bp); there is very little expression during growth. Induction during development relies on at least two positive regulatory elements located in the coding region of the next gene upstream. At least two positive regulatory elements and one negative element lie downstream of the dev promoter, such that the region controlling dev expression spans more than 1 kb. The results of testing different fragments for dev promoter activity in wild-type and devS mutant backgrounds strongly suggest that upstream and downstream regulatory elements interact functionally. Strikingly, the 37-bp sequence between the two CRISPR repeats that, minimally, are cotranscribed with dev and cas genes exactly matches a sequence in the bacteriophage Mx8 intP gene, which encodes a form of the integrase needed for lysogenization of M. xanthus.

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Independent regulation of H-NS-mediated silencing of the bgl operon at two levels: upstream by BglJ and LeuO and downstream by DnaKJ

Microbiology ◽

10.1099/mic.0.28080-0 ◽

2005 ◽

Vol 151 (10) ◽

pp. 3349-3359 ◽

Cited By ~ 24

Author(s):

S. Madhusudan ◽

Andreas Paukner ◽

Yvonne Klingen ◽

Karin Schnetz

Keyword(s):

Escherichia Coli ◽

Transcription Initiation ◽

Promoter Activity ◽

Transposon Mutagenesis ◽

Open Reading Frames ◽

Wild Type ◽

Coding Region ◽

First Time ◽

Insertion Mutations ◽

Reading Frames

Silencing of the Escherichia coli bgl operon by the histone-like nucleoid-structuring protein H-NS occurs at two levels. Binding of H-NS upstream of the promoter represses transcription initiation, whilst binding within the coding region is also proposed to repress transcription elongation. The latter, downstream level of repression is counteracted by the protease Lon and, thus, silencing of the bgl operon is more effective in lon mutants. Transposon-mutagenesis screens for suppression of this lon phenotype on bgl were performed and insertion mutations disrupting rpoS and crl were obtained, as well as mutations mapping upstream of the open reading frames of bglJ, leuO and dnaK. In rpoS and crl mutants, bgl promoter activity is known to be higher. Likewise, as shown here, bgl promoter activity is increased in the bglJ and leuO mutants, which express BglJ and LeuO constitutively. However, BglJ and LeuO have no impact on downstream repression. A dnaKJ mutant was isolated for the first time in the context of the bgl operon. The mutant expresses lower levels of DnaK than the wild-type. Interestingly, in this dnaKJ : : miniTn10 mutant, downstream repression of bgl by H-NS is less effective, whilst upstream repression by H-NS remains unaffected. Together, the data show that the two levels of bgl silencing by H-NS are regulated independently.

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