Protein/RNA interaction in light regulated translation in the chloroplast (original) (raw)
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Genes & Development, 1993
The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known. In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion. Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments. Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp. By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations. In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn o...
HMG1 proteins from evolutionary distant organisms distort B-DNA conformation in similar way
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1999
The abundant high-mobility group proteins 1/2 (HMG1/2) represent a group of potent architectural elements of chromatin. They are able to induce strong bends and untwist DNA. Here, we compared the abilities of diverse HMG1 proteins to distort the B-DNA conformation of 30-base pair DNA fragment. The DNA bending was measured in solution by monitoring fluorescence resonance energy transfer between fluorescence probes attached to opposite ends of the DNA fragment. Various insect and plant proteins which differ in size, in composition of their HMG1-box domains (HMG1-BD), and in composition of the N-and the C-terminally flanking regions were analyzed in these experiments. Despite these structural differences the extent of the induced changes in DNA conformation upon binding to various proteins was similar, as the estimated bend angle was 150 þ 20³ for all the tested proteins. Our results suggest that a set of highly conserved residues stabilizing the tertiary structure of the HMG1-BD mainly determines the extent of DNA bending in the complex. Even extended positively charged regions flanking the HMG1-BD are apparently not able to influence this conformational distortion of DNA.
Proceedings of The National Academy of Sciences, 2007
A salient feature of organelle gene expression is the requirement for nucleus-encoded factors that act posttranscriptionally in a gene-specific manner. A central issue is to understand whether these factors are merely constitutive or have a regulatory function. In the unicellular alga Chlamydomonas reinhardtii, expression of the chloroplast petA gene-encoding cytochrome f, a major subunit of the cytochrome b 6f complex, depends on two specific nucleusencoded factors: MCA1, required for stable accumulation of the petA transcript, and TCA1, required for its translation. We cloned the TCA1 gene, encoding a pioneer protein, and transformed appropriate mutant strains with tagged versions of MCA1 and TCA1. In transformed strains expressing decreasing amounts of MCA1 or TCA1, the concentration of these factors proved limiting for petA mRNA accumulation and cytochrome f translation, respectively. This observation suggests that in exponentially growing cells, the abundance of MCA1 sets the pool of petA transcripts, some of which are TCA1-selected for an assembly-dependent translation of cytochrome f. We show that MCA1 is a short-lived protein. Its abundance varies rapidly with physiological conditions that deeply affect expression of the petA gene in vivo, for instance in aging cultures or upon changes in nitrogen availability. We observed similar but more limited changes in the abundance of TCA1. We conclude that in conditions where de novo biogenesis of cytochrome b 6f complexes is not required, a rapid drop in MCA1 exhausts the pool of petA transcripts, and the progressive loss of TCA1 further prevents translation of cytochrome f.
Journal of Biological Chemistry
The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single-or double-strand^ DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to singlestranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 m M Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.
Post-translational modifications of non-histone high mobility group nucleosomal proteins
2007
High mobility group nucleosomal proteins, HMGN-1 and HMGN-2, regulate several DNAdependent viz., transcription and replication activities of the mammalian nucleus. HMGN-1 decondensates the nucleosome, when it interferes with the linkage of histones to DNA. The HMGN-1 targets histones like H2B, H3 and linker histone H1. The histones-DNA interference is regulated by post-translational modifications at the Ser/Thr/Tyr residues of HMGN-1. Phosphorylation of HMGN-1 inhibits replication/transcription, whereas O-GlcNAc modification of HMGN-1 promotes replication/transcription. This in silico study proposes that, phosphorylation sites at Ser/Thr residues also exhibit O-GlcNAc modification potential. The prediction of O-GlcNAc modification sites and possible interplay between phosphorylation and O-glycosylation will enhance understanding of gene regulation.
RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1
Nucleic Acids Research, 2000
The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5'-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5'-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine-Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.