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David L Herrin

Publications and source records attributed to David L Herrin.

13 recordsLinked to original sources
PubMed2006

Distinct roles for the 5' and 3' untranslated regions in the degradation and accumulation of chloroplast tufA mRNA: identification of an early intermediate in the in vivo degradation pathway.

Elongation factor Tu in Chlamydomonas reinhardtii is a chloroplast-encoded gene (tufA) whose 1.7-kb mRNA has a relatively short half-life. In the presence of chloramphenicol (CAP), which freezes translating chloroplast ribosomes, a 1.5-kb tufA RNA becomes prominent. Rifampicin-chase analysis indicates that the 1.5-kb RNA is a degradation intermediate, and mapping studies show that it is missing 176-180 nucleotides from the 5' end of tufA. The 5' terminus of the intermediate maps to a section of the untranslated region (UTR) predicted to be highly structured and to encode a small ORF. The intermediate could be detected in older cultures in the absence of CAP, indicating that it is not an artifact of drug treatment. Also, it did not overaccumulate in the chloroplast ribosome-deficient mutant, ac20 cr1, indicating its stabilization is specific to elongation-arrested ribosomes. To determine if the 5' UTR of tufA is destabilizing, the corresponding region of the atpA-aadA-rbcL gene was replaced with the tufA sequence, and introduced into the chloroplast genome; the 3' UTR was also substituted for comparison. Analysis of these transformants showed that the transcripts containing the tufA 3'-UTR accumulate to significantly lower levels. Data from constructs based on the vital reporter, Renilla luciferase, confirmed the importance of the tufA 3'-UTR in determining RNA levels, and suggested that the 5' UTR of tufA affects translation efficiency. These data indicate that the in vivo degradation of tufA mRNA begins in the 5' UTR, and is promoted by translation. The data also suggest, however, that the level of the mature RNA is determined more by the 3' UTR than the 5' UTR.

3' Untranslated Regions↗
PubMed2006

Mg2+ mimicry in the promotion of group I ribozyme activities by aminoglycoside antibiotics.

Aminoglycoside antibiotics inhibit several types of ribozymes, including group I introns, by displacing critical Mg2+ ions. However, they stimulate activity of the small hairpin ribozyme. We show here that aminoglycosides promote self-splicing of the Cr.psbA2 group I intron at subthreshold Mg2+ concentrations. Neomycin is the most effective of the aminoglycosides tested; it stimulates splicing of Cr.psbA2 at micromolar concentrations, and, in this respect, is >100-fold more effective than spermidine. At optimal Mg2+ for Cr.psbA2 splicing, these drugs, especially kanamycin B and tobramycin, promote GTP attack at the 3' splice-site. Kinetic analysis suggests that this is due to an alternatively folded state of the ribozyme that is induced, or stabilized, by aminoglycosides. A similar effect is observed at high Mg2+ concentrations. Comparing the effects of structurally related aminoglycosides indicates that splicing promotion is more sensitive to drug structure than misfolding and occurs at lower drug concentrations. These data show that aminoglycosides can promote biochemical activities of a large ribozyme by acting as a Mg2+ mimic. The results also underscore the functional diversity of group I introns in nature.

Aminoglycosides↗
PubMed2006

Unusual metal specificity and structure of the group I ribozyme from Chlamydomonas reinhardtii 23S rRNA.

Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg2+ and Mn2+ support both processes. However, some group I introns are active only with Mg2+, e.g. three of the five group I introns in Chlamydomonas reinhardtii. We have investigated one of these ribozymes, an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii (Cr.LSU), by determining if the inhibition by Mn2+ involves catalysis, folding, or both. Kinetic analysis of guanosine-dependent cleavage by a Cr.LSU ribozyme, 23S.5 Delta Gb, that lacks the 3' exon and intron-terminal G shows that Mn2+ does not affect guanosine binding or catalysis, but instead promotes misfolding of the ribozyme. Surprisingly, ribozyme misfolding induced by Mn2+ is highly cooperative, with a Hill coefficient larger than that of native folding induced by Mg2+. At lower Mn2+ concentrations, metal inhibition is largely alleviated by the guanosine cosubstrate (GMP). The concentration dependence of guanosine cosubstrate-induced folding suggests that it functions by interacting with the G binding site, perhaps by displacing an inhibitory Mn2+. Because of these and other properties of Cr.LSU, the tertiary structure of the intron from 23S.5 Delta Gb was examined using Fe2+-EDTA cleavage. The ground-state structure shows evidence of an unusually open ribozyme core: the catalytic P3-P7 domain and the nucleotides that connect it to the P4-P5-P6 domain are exposed to solvent. The implications of this structure for the in vitro and in vivo properties of this intron ribozyme are discussed.

Animals↗
PubMed2005

Expression, purification, and biochemical characterization of the intron-encoded endonuclease, I-CreII.

The ORF of the Cr.psbA4 intron of Chlamydomonas reinhardtii mediates efficient intron homing, and contains an H-N-H and possibly a GIY-YIG motif. The ORF was over-expressed in Escherichia coli without non-native amino acids, but was mostly insoluble. However, co-over-expression of E. coli chaperonins GroEL/GroES solubilized approximately 50% of the protein, which was purified by ion-exchange and heparin-affinity chromatography. Biochemical characterization showed that the protein is a double-strand-specific endonuclease that cleaves fused psbA exon 4-exon 5 DNA, and was named I-CreII. I-CreII has a relatively relaxed divalent metal ion requirement (Mg(2+), Mn(2+), Ca(2+), and Fe(2+) supported cleavage), is insensitive to salt <350 mM, and is stabilized by DNA. Cleavage of target DNA occurs close (4 nt on the top strand) to the intron-insertion site, and leaves 2-nt 3'-OH overhangs, similar to GIY-YIG endonucleases. The boundaries of the recognition sequence span approximately 30 bp, and encompass the cleavage and intron-insertion sites. Cleavage of heterologous psbA DNAs indicates the enzyme can tolerate multiple, but not all, substitutions in the recognition site. This work will facilitate further study of this novel endonuclease, which may also find use in site-specific manipulation of chloroplast DNA.

Algal Proteins↗
PubMed2004

Chloroplast RNA processing and stability.

Primary chloroplast transcripts are processed in a number of ways, including intron splicing, internal cleavage of polycistronic RNAs, and endonucleolytic or exonucleolytic cleavages at the transcript termini. All chloroplast RNAs are also subject to degradation, although a curious feature of many chloroplast mRNAs is their relative longevity. Some of these processes, e.g., psbA splicing and stability of a number of chloroplast mRNAs, are regulated in response to light-dark cycles or nutrient availability. This review highlights recent advances in our understanding of these processes in the model organism Chlamydomonas reinhardtii, focusing on results since the extensive reviews published in 1998 [Herrin DL et al. 1998 (pp. 183-195), Nickelsen Y 1998 (pp. 151-163), Stern DB and Drager RG 1998 (pp. 164-182), in Rochaix JD et al. (eds) The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands]. We also allude to studies with other organisms, and to the potential impact of the Chlamydomonas genome project where appropriate.

Journal Article↗
PubMed2004

Structure, circadian regulation and bioinformatic analysis of the unique sigma factor gene in Chlamydomonas reinhardtii.

In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded bacterial RNAP in which promoter specificity is conferred by nuclear-encoded sigma factors, and a nuclear-encoded phage-like RNAP. Green algae, however, appear to possess only the bacterial enzyme. Since transcription of much, if not most, of the chloroplast genome in Chlamydomonas reinhardtii is regulated by the circadian clock and the nucleus, we sought to identify sigma factor genes that might be responsible for this regulation. We describe a nuclear gene (RPOD) that is predicted to encode an 80 kDa protein that, in addition to a predicted chloroplast transit peptide at the N-terminus, has the conserved motifs (2.1- 4.2) diagnostic of bacterial sigma-70 factors. We also identified two motifs not previously recognized for sigma factors, adjacent PEST sequences and a leucine zipper, both suggested to be involved in protein-protein interactions. PEST sequences were also found in approximately 40% of sigma factors examined, indicating they may be of general significance. Southern blot hybridization and BLAST searches of the genome and EST databases suggest that RPODmay be the only sigma factor gene in C. reinhardtii. The levels of RPODmRNA increased 2- 3-fold in the mid-to-late dark period of light-dark cycling cells, just prior to, or coincident with, the peak in chloroplast transcription. Also, the dark-period peak in RPOD mRNA persisted in cells shifted to continuous light or continuous dark for at least one cycle, indicating that RPODis under circadian clock control. These results suggest that regulation of RPODexpression contributes to the circadian clock's control of chloroplast transcription.

Journal Article↗
PubMed2004

A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self-splicing and genetic evidence for maturase activity.

The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp. from Antarctica that self-splices accurately in vitro. Remarkably, this intron, which also encodes an ORF with putative reverse transcriptase, maturase, and endonuclease domains, is in the same location, and is related to the E. myxocylindracea intron, as well as to group IIB2 introns from cyanobacteria. In vitro self-splicing of Chs.psbA1 occurred via a lariat, and required Mg(2+) (>12 mM) and NH(4)(+). Self-splicing was improved by deleting most of the ORF and by using pre-RNAs directly from transcription reactions, suggestive of a role for folding during transcription. Self-splicing of Chs.psbA1 pre-RNAs showed temperature optima of ~44 degrees C, but with a broad shoulder on the low side of the peak; splicing was nearly absent at 50 degrees C, indicative of thermolability. Splicing of wild-type Chs.psbA1 also occurred in Escherichia coli, but not when the ORF was disrupted by mutations, providing genetic evidence that it has maturase activity. This work provides the first description of a ribozyme from a psychrophilic organism. It also appears to provide a second instance of interkingdom horizontal transfer of this group IIB2 intron (or a close relative) from cyanobacteria to chloroplasts.

Animals↗
PubMed2003

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth.

The chloroplast-encoded psbA gene encodes the D1 polypeptide of the photosystem II reaction center, which is synthesized at high rates in the light. In Chlamydomonas reinhardtii, the psbA gene contains four self-splicing group I introns whose rates of splicing in vivo are increased at least 6-10-fold by light. However, because psbA is an abundant mRNA, and some chloroplast mRNAs appear to be in great excess of what is needed to sustain translation rates, the developmental significance of light-promoted splicing has not been clear. To address this and other questions, potentially destabilizing substitutions were made in several predicted helices of the fourth psbA intron, Cr.psbA4, and their effects on in vitro and in vivo splicing assessed. Two-nucleotide substitutions in P4 and P7 were necessary to substantially reduce splicing of this intron in vivo, although most mutations reduced self-splicing in vitro. The P7-4,5 mutant, whose splicing was completely blocked, showed no photoautotrophic growth and synthesis of a truncated D1 (exons 1-4) polypeptide from the unspliced mRNA. Most informative was the P4'-3,4 mutant, which exhibited a 45% reduction in spliced psbA mRNA, a 28% reduction in synthesis of full-length D1, and an 18% reduction in photoautotrophic growth. These results indicate that psbA mRNA is not in great excess, and that highly efficient splicing of psbA introns, which is afforded by light conditions, is necessary for optimal photosynthetic growth.

Animals↗
PubMed2003

A cosmid vector containing a dominant selectable marker for cloning Chlamydomonas genes by complementation.

Cosmid vectors containing a dominate selectable marker (ble) for complementation cloning of genes in Chlamydomonas reinhardtii were created. The usefulness of these vectors, which differ in the orientation of the ble cassette, was demonstrated by transforming C. reinhardtii to phleomycin resistance, by constructing a large library (approximately 5 x 10(5) recombinants) in one of them using DNA from a C. reinhardtii mutant, and by transforming C. reinhardtii with recombinant cosmid clones and pools.

Animals↗
PubMed2002

FUGOID: functional genomics of organellar introns database.

FUGOID is a web-based, taxonomically broad organelle intron database that collects and integrates various functional and structural data on organellar (mitochondrial and chloroplast) introns. The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism. It is implemented in a relational database management system, allowing sophisticated, user-friendly searching, data entry and revision. Users can access the database by any common web browser using a variety of operating systems. The main page of the database is available at http://wnt.cc.utexas.edu/~ifmr530/introndata/main.htm.

Animals↗
PubMed2002

Assessing the relative importance of light and the circadian clock in controlling chloroplast translation in Chlamydomonas reinhardtii.

Previous work has shown that transcription of a number of chloroplast-encoded genes, including those for photosynthesis, are under circadian clock control in Chlamydomonas reinhardtii. However, some of these genes encode long-lived mRNAs that are also subject to translational control. Rates of synthesis of the major chloroplast translation products vary dramatically (10-20-fold) during light-dark (LD) cycles, peaking in the light period. To determine whether this pattern reflects circadian clock control, LD-grown cells were shifted to continuous light (LL) and chloroplast protein synthesis monitored by periodic pulse-labeling in the presence of cycloheximide; chloroplast protein synthesis in LD was also examined for comparison. The LD patterns of synthesis of the major polypeptides (including D1, D2, and the large subunit of ribulose-1,5-bisphosphate carboxylase (LS)) were similar to those obtained previously in the absence of cycloheximide. In the LL condition, rates of synthesis of the major chloroplast translation products were high throughout the period examined ( approximately 36 h), fluctuating > 3-fold, although they were generally higher in the subjective light period. LD-grown cells were also shifted to continuous dark (DD) and chloroplast protein synthesis analyzed for approximately 24 h starting from the mid-dark period. There was a gradual decline in synthesis of the major proteins during the first subjective light period, which was followed by a very small peak in synthesis around the second subjective dark --> light transition. RNA blot analysis showed that the mRNAs for D1, D2 and LS were present at high levels during the period of declining translation. These results indicate that with photoautotrophic growth in LD cycles, the illumination conditions per se are more important than the clock in determining chloroplast translation, but the clock may contribute to this regulation. The advantages of controlling translation by a direct light response and transcription primarily by the circadian clock are discussed. Finally, evidence of translational control of elongation factor Tu synthesis was obtained.

Journal Article↗
PubMed2002

Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii.

Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the chloroplast group I rRNA intron of Chlamydomonas reinhardtii, Cr.LSU. In vitro assays showed that these substitutions had surprisingly strong effects on Cr.LSU self-splicing; however, splicing of all but the P6 mutations could be at least partially recovered by increasing the Mg2+ concentration. The mutant constructs were transformed into chloroplasts to replace the wild-type intron; however, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-deficient P4125A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least two nuclear genes. The 7151 suppressor mutation, which defines the chloroplast-splicing suppressor (css1) gene, had no obviously altered growth phenotype with the wild-type intron, and was dominant in vegetative diploids containing the mutant intron. All three of the suppressor strains also suppressed a mutation in the P4 region of the fourth psbA intron, Cr.psbA4, indicating that these genes play a role in splicing of multiple group I introns in the chloroplast.

Animals↗