Friedrich Buck

1H NMR study of the lactose repressor from Escherichia coli

Although many investigations have been carried out on the functional properties of the Zuc repressor of Escherichia coli using biochemical or genetic methods (reviewed [ 1,2]) only few attempts have been made to elucidate the structure of this protein at the molecular level with physical methods. X-ray analysis of the tertiary structure has not yet been successful. “F NMR studies have been performed with a protein derivative containing only [3-1gF]tyrosine residues instead of normal tyrosines [3]. A similar ‘H NMR investigation of the tyrosine residues has been described [4]. The ‘H NMR spectra were simplified by growing a special E. coli strain on a mixture of deuterated amino acids and [2,6-‘Hltyrosine. Hence all the ring proton resonances except the tyrosine 3,5-proton resonances have disappeared from the spectrum. In order to analyse the interaction of the operatorDNA binding site of the Zac repressor which is constituted of the amino-terminal 60 residues (reviewed [ 1,5]) with the aggregating and inducer binding part of the Zuc repressor [6] we have studied the tryptic digestion products of native Zac repressor [7] with ‘H NMR and compared the corresponding spectra with those of the native protein. We present evidence in favour of an identical and defined tertiary structure for both the amino-terminal region of the luc repressor and the corresponding dissected monomeric amino-terminal tryptic fragments [8] accounting for residues l-5 1 and l-59 of the Zac repressor [7]. It is also suggested that this amino-terminal domain is linked to a rigid repressor core by a region of surprisingly high flexibility [8].

Interaction of brain somatostatin receptors with the PDZ domains of PSD-95

Using peptide affinity purification, we identified an interaction between somatostatin receptors SSTR4 and SSTR1 and PDZ domains 1 and 2 of the postsynaptic proteins postsynaptic density protein of 95 kDa (PSD‐95) and PSD‐93. The existence of the SSTR4/PSD‐95 complex was verified by coimmunoprecipitation from transfected cells and solubilized brain membranes. In neurons, dendritically localized SSTR4 partially colocalizes with postsynaptic PSD‐95. As functional parameters of the receptor, such as coupling to potassium channels, are not affected by the interaction with PSD‐95, the association may serve to localize the receptor to postsynaptic sites.

A non-canonical initiation site is required for efficient translation of the dendritically localized Shank1 mRNA.

Local protein synthesis in dendrites enables neurons to selectively change the protein complement of individual postsynaptic sites. Though it is generally assumed that this mechanism requires tight translational control of dendritically transported mRNAs, it is unclear how translation of dendritic mRNAs is regulated. We have analyzed here translational control elements of the dendritically localized mRNA coding for the postsynaptic scaffold protein Shank1. In its 5′ region, the human Shank1 mRNA exhibits two alternative translation initiation sites (AUG+1 and AUG+214), three canonical upstream open reading frames (uORFs1-3) and a high GC content. In reporter assays, fragments of the 5′UTR with high GC content inhibit translation, suggesting a contribution of secondary structures. uORF3 is most relevant to translation control as it overlaps with the first in frame start codon (AUG+1), directing translation initiation to the second in frame start codon (AUG+214). Surprisingly, our analysis points to an additional uORF initiated at a non-canonical ACG start codon. Mutation of this start site leads to an almost complete loss of translation initiation at AUG+1, demonstrating that this unconventional uORF is required for Shank1 synthesis. Our data identify a novel mechanism whereby initiation at a non-canonical site allows for translation of the main Shank1 ORF despite a highly structured 5′UTR.

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator OR3 oligonucleotide, which is the preferential binding site for the Cro repressor of phage λ, was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1′ and the H2′-H2″ sugar protons of the OR3 operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the OR3 operator — Cro repressor interaction.

NMR Investigation of Palindromic LAC Operator DNA Sequences of Varying Size: Influences of Complex Formation with the LAC Repressor Headpiece on the DNA 1H Resonances

Abstract 1H-NMR studies on s3rmmetrical lac operators were performed to determine the minimum size of a lac operator for tight complex formation with the lac repressor headpiece. Four operators of varying size from 18 base pairs to 24 base pairs were chemically synthesized. The data obtained suggest that for a synthetic lac operator to form a tight complex with the lac headpiece it should be at least 22 base pairs long.