Acquisition of myogenic specificity through replacement of one amino acid of MASH-1 and introduction of an additional alpha-helical turn.

The homologous transcription factors Myf-5, MyoD, myogenin, MRF-4, and MASH-1 bind with high affinity and modest sequence specificity to DNA containing an E-box (CANNTG). This similarity of the in vitro DNA binding specificity is in sharp contrast to the high physiological specificity displayed by these proteins. Myf-5, MyoD, myogenin, and MRF-4 induce cells to differentiate along a myogenic pathway, while MASH-1 promotes the differentiation of neuronal precursor cells. We show here that MASH-1 can be converted into a protein capable of inducing myogenesis in fibroblasts by replacing leucine (130) of MASH-1 with lysine and introducing an additional turn into its basic recognition helix. These changes do not significantly alter the DNA binding properties of the proteins in cell free conditions. Crystallographic data for the DNA complexes of MyoD and E12 suggest that Leu (130) points away from the DNA into the solvent. We postulate that the identity of the amino acid in position 130 is important for protein-protein interactions that might affect the DNA binding specificities displayed by BHLH-proteins in vivo and form the molecular basis of the different physiological properties of the myogenic and neurogenic BHLH-proteins.

Circadian synthesis of light-harvesting-chlorophyll-proteins in Euglena gracilis is under translational control.

Two proteins with apparent molecular masses of 17 and 24 kD that are synthesized in a circadian manner in the phytoflagellate Euglena gracilis, were recognized as proteins belonging to the family of light-harvesting-chlorophyll-proteins (LHCPs) of class I (17 kD) and of class II (24 kD). Identification was achieved by N-terminal sequencing of the proteins isolated from two-dimensional polyacrylamide gels and by detection with an anti-LHCP II serum. While it was found that the total amount of LHCPs remains almost constant, when Euglena is grown under diurnal conditions (12 h light and 12 h dark), we could show that the amount of newly synthesized 17 and 24 kD proteins varies about 20-fold with a maximum of synthesis in the light phase. In contrast, the analysis of the mRNA levels at different times revealed only minor differences in the stationary concentration of the LHCP specific mRNA, indicating that the control of LHCP synthesis is at the translational level. Principally, the same finding was obtained using inhibitors of transcription. Thus, it is concluded that the expression of LHCPs in Euglena gracilis in contrast to that of higher plants is primarily regulated at the translational level.

Characterization of polypeptides in Euglena gracilis which are synthesized in a circadian manner.

In vivo labeling of proteins in the unicellular phytoflagellate Euglena gracilis, grown under constant light, with [35S]methionine, gave evidence that three proteins of 17,000, 24,000 and 60,000 M(r) were synthesized rhythmically with a period length of 26 h. The 60,000 M(r) protein was synthesized at the subjective end of the 12 h light phase, while the two smaller proteins of 17,000 and 24,000 M(r) shared a maximum of synthesis at the subjective noon. The rate of synthesis oscillated up to 20-fold when the maxima and minima were compared. By isoelectric focusing, the 24,000 M(r) protein was separated into three distinct spots. When different temperatures between 16 and 27 degrees C were applied, the period length of the circadian synthesis rhythms differed by only 3 to 5 h, indicating that the synthesis rhythms of these proteins were almost temperature compensated. Synthesis of the two smaller peptides was inhibited by cycloheximide but not by chloramphenicol, implying that synthesis occurred at 80S ribosomes. Once synthesized and processed, these two proteins were fairly stable in continuous light for about 120 h, while they were degraded during darkness- although slowly. After cell breakage, these proteins were localized in the pelletable membranous fraction.

Covalently linking BHLH subunits of MASH-1 increases specificity of DNA binding.

MASH-1, a member of the basic-helix-loop-helix (BHLH) family of transcription factors, promotes the differentiation of committed neuronal precursor cells. In vitro, MASH-1 displays only marginal DNA sequence specificity. We have produced a MASH-1 variant, MASH-GGC, by introducing the tripeptide Gly-Gly-Cys at the C-terminal end of the BHLH domain. Under reducing conditions the properties of MASH-GGC and of the BHLH domain of MASH-1 were very similar. Like MASH-1, reduced MASH-GGC showed little specificity of DNA binding. CD spectroscopy revealed that both proteins underwent a conformational change from a largely unfolded to a mainly alpha-helical conformation upon binding to DNA. When the subunits of MASH-GGC were linked through a disulfide bond, the folded conformation was stable over a wide concentration range (2.5 nM to 2 microM) even in the absence of DNA. Oxidized MASH-GGC bound to E-box-containing sequences half-maximally at 148 nM, compared to 458 nM for the reduced form. Therefore, even when the change from a monomeric to a dimeric species was taken into account, the affinity for E-box-containing DNA sequences was increased. Surprisingly, the apparent dissociation constant for the complex with DNA not containing E-box sequences was increased upon oxidation. Therefore, despite the large distance between the disulfide bridge and the protein-DNA interface, covalently linking the subunits of MASH-1 increased the specificity of DNA binding significantly. In vivo, such an increase of the intrinsic DNA binding specificity might be achieved through interactions with other proteins of the transcriptional machinery.

Basic helix-loop-helix protein MyoD displays modest DNA binding specificity.

The expression of MyoD can activate muscle specific genes and myogenic differentiation in many cell types. The hypothesis that the DNA binding specificity of MyoD is responsible for its biological specificity was tested. Homodimers of MyoD bind to E-box containing DNA with high affinity, but do not form stable and well defined complexes with heterologous DNA sequences. The physiologically active heterodimer of MyoD and E12 binds an oligonucleotide containing an E-box sequence with an affinity only two orders of magnitude higher than a completely unrelated DNA sequence, stressing the importance of cooperative interactions with other proteins of the transcriptional machinery for specific gene activation.