Light-regulated translation of chloroplast proteins. I. Transcripts of psaA-psaB, psbA, and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings

We have previously observed (Klein, R. R., and J. E. Mullet, 1986, J. Biol. Chem. 261:11138-11145) that translation of two 65-70-kD chlorophyll a-apoproteins of Photosystem I (gene products of psaA and psaB) and a 32-kD quinone-binding protein of Photosystem II (gene product of psbA) was not detected in plastids of dark-grown barley seedlings even though transcripts for these proteins were present. In the present study it was found that nearly all of the psaA-psaB transcripts in plastids of dark-grown plants were associated with membrane-bound polysomes. Membrane-associated polysomes from plastids of dark-grown plants synthesized the 65-70-kD chlorophyll a-apoproteins at low levels when added to a homologous in vitro translation extract capable of translation elongation. However, when etioplast membranes were disrupted with detergent, in vitro synthesis of the 65-70-kD chlorophyll a-apoproteins increased to levels observed with polysomes of plastids from illuminated plants. These results suggest that synthesis of the chlorophyll a-apoproteins of Photosystem I is arrested on membrane-bound polysomes at the level of polypeptide chain elongation. In addition to the selective activation of chlorophyll a-apoprotein translation, illumination also caused an increase in chloroplast polysomes (membrane-associated and stromal) and induced a recruitment of psbA and rbcL transcripts into chloroplast polysomes. These results indicate that in conjunction with the selective activation of chlorophyll a-apoprotein elongation, illumination also caused a general stimulation of chloroplast translation initiation.

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In Vitro Synthesis, Transport, and Assembly of the Constituent Polypeptides of the Light-Harvesting Chlorophyll a/b Protein Complex

Genome Organization and Expression in Plants ◽

10.1007/978-1-4613-3051-6_29 ◽

1980 ◽

pp. 337-351 ◽

Cited By ~ 26

Author(s):

Gregory W. Schmidt ◽

Sue Bartlett ◽

Arthur R. Grossman ◽

Anthony R. Cashmore ◽

Nam-Hai Chua

Keyword(s):

Chlorophyll A ◽

Protein Complex ◽

Light Harvesting ◽

In Vitro Synthesis

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Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

Biochemistry and Cell Biology ◽

10.1139/o91-074 ◽

1991 ◽

Vol 69 (8) ◽

pp. 499-508 ◽

Cited By ~ 36

Author(s):

Andrea G. Bodnar ◽

Richard A. Rachubinski

Keyword(s):

Endoplasmic Reticulum ◽

Immunoblot Analysis ◽

In Vitro Translation ◽

Peroxisome Proliferator ◽

Peroxisomal Membrane ◽

Molecular Masses ◽

Fold Induction ◽

Membrane Bound ◽

Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

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Probing in vitro translation products with monoclonal antibodies to a 15.2 kD polypeptide subunit of photosystem I

Carlsberg Research Communications ◽

10.1007/bf02907147 ◽

1985 ◽

Vol 50 (4) ◽

pp. 211-221 ◽

Cited By ~ 14

Author(s):

Gunilla Høyer-Hansen ◽

Lisbeth Skou Hønberg ◽

Peter Bordier Høj

Keyword(s):

Monoclonal Antibodies ◽

Photosystem I ◽

In Vitro Translation

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Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis

Journal of Virology ◽

10.1128/jvi.73.12.10104-10112.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10104-10112 ◽

Cited By ~ 98

Author(s):

David J. Barton ◽

B. Joan Morasco ◽

James B. Flanegan

Keyword(s):

Protein Synthesis ◽

Polypeptide Chain ◽

Rna Replication ◽

Viral Rna ◽

In Vitro Translation ◽

Chain Elongation ◽

Protein Synthesis Inhibitors ◽

Positive Strand ◽

Negative Strand

ABSTRACT Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation “freeze” ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.

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Reconstitution of yeast translation elongation and termination in vitro utilizing CrPV IRES-containing mRNA

The Journal of Biochemistry ◽

10.1093/jb/mvaa021 ◽

2020 ◽

Vol 167 (5) ◽

pp. 441-450

Author(s):

Taisho Abe ◽

Riku Nagai ◽

Hiroaki Imataka ◽

Nono Takeuchi-Tomita

Keyword(s):

Molecular Weight ◽

Translation Initiation ◽

In Vitro Translation ◽

Translation System ◽

Reporter Protein ◽

Translation Elongation ◽

Initiation Factors ◽

Translation Factors ◽

Internal Translation

Abstract We developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors and programmed by CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing the active reporter protein, nanoLuciferase, with a molecular weight of 19 kDa. The protein synthesis by the system is appropriately regulated by controlling its composition, including translation factors, amino acids and antibiotics. We found that a high eEF1A concentration relative to the ribosome concentration is critically required for efficient IRES-mediated translation initiation, to ensure its dominance over IRES-independent random internal translation initiation.

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Bovine beta 1----4-galactosyltransferase: two sets of mRNA transcripts encode two forms of the protein with different amino-terminal domains. In vitro translation experiments demonstrate that both the short and the long forms of the enzyme are type II membrane-bound glycoproteins.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)39770-4 ◽

1990 ◽

Vol 265 (6) ◽

pp. 3324-3331

Author(s):

R N Russo ◽

N L Shaper ◽

J H Shaper

Keyword(s):

In Vitro Translation ◽

Type Ii ◽

Amino Terminal ◽

Mrna Transcripts ◽

Membrane Bound ◽

Terminal Domains

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Transcriptional and translational mechanisms of cytochrome b5 reductase isoenzyme generation in humans

Biochemical Journal ◽

10.1042/bj3550529 ◽

2001 ◽

Vol 355 (2) ◽

pp. 529-535 ◽

Cited By ~ 7

Author(s):

Alena LEROUX ◽

Luisa MOTA VIEIRA ◽

Axel KAHN

Keyword(s):

Cytochrome B5 ◽

Protein Isoforms ◽

In Vitro Translation ◽

Alu Elements ◽

Cytochrome B5 Reductase ◽

Dynamic Events ◽

Membrane Bound ◽

Essential Enzyme ◽

Translational Mechanisms

Cytochrome b5 reductase (b5R) is an essential enzyme that exists in soluble and membrane-bound isoforms, each with specific functions. In the rat, the two forms are generated from alternative transcripts differing in the first exons. In contrast, the biogenesis of b5R isoforms in the human is not yet well understood. In the present study we have detected three novel alternative exons, designated 1S, S′ and 1B, located between the first alternative exon 1M and the common second exon in the human b5R gene. Accordingly, multiple M-type, S-type and SS′-type and B-type transcripts are generated. All types of human b5R transcript are expressed ubiquitously. An analysis of in vitro translation products demonstrated an alternative use of different AUG initiators resulting in the production of various human b5R protein isoforms. Our results indicate that the organization of the 5′ region of the b5R gene is not conserved between rodents and humans. Insertion of Alu elements into the human b5R gene, in particular just upstream of the S/S′ region, could be responsible for dynamic events of gene rearrangement during evolution.

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In vitro synthesis of a brain-specific protein (S-100) by free and membrane-bound polysomes

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis ◽

10.1016/0005-2787(73)90236-0 ◽

1973 ◽

Vol 299 (4) ◽

pp. 634-641 ◽

Cited By ~ 10

Author(s):

Paola Amaldi ◽

Graziella Rusca ◽

Pietro Calissano

Keyword(s):

Protein S ◽

Specific Protein ◽

In Vitro Synthesis ◽

S 100 ◽

Membrane Bound

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In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling

Methods and Protocols ◽

10.3390/mps4030045 ◽

2021 ◽

Vol 4 (3) ◽

pp. 45

Author(s):

Riku Nagai ◽

Yichen Xu ◽

Chang Liu ◽

Ayaka Shimabukuro ◽

Nono Takeuchi-Tomita

Keyword(s):

Unnatural Amino Acids ◽

Molecular Mechanisms ◽

In Vitro Translation ◽

Critical Parameters ◽

Translation System ◽

Reaction Conditions ◽

Translation Elongation ◽

Functional Studies ◽

Ribosome Stalling

The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.

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