1H-NMR study of the lambda operator site OL1: assignment of the imino and adenine H2 resonances.

One- and two-dimensional proton NMR methods are being used to study the synthetic lambda operator site O-L1, a 17 base-pair DNA duplex recognized by lambda repressor and Cro protein. The complete assignment of the 17 imino protons, which participate in Watson-Crick hydrogen bonding, and of the eight adenine H2 protons, which lie in the minor groove of the double helix, is presented.     

Behavior of the imino protons of the lambda OR3 17mer studied by high resolution NMR

Imino proton resonances of lambda OR3 17mer were observed with time-shared Redfield pulse method by using a JEOL 500 MHz NMR instrument. They show gradual broadening and disappearance with the elevation of temperature indicating the stepwise melting of the duplex. By the selective irradiation at each peak position nuclear Overhauser effects were observed between the imino and adenine C2H protons and between imino protons themselves. Combining these data, fifteen imino proton resonances could be assigned to each base pair except two terminal AT base pairs. Based on the assignment it can be said that AT rich regions near the terminal melt first, followed by the melting of the inside GC rich core. The two AT base pairs in the middle of the GC core are resistant to heat. Spin lattice relaxation times were also observed and the results are consistent with the melting profile.     

Sequence-specific recognition of deoxyribonucleic acid. Chemical synthesis and nuclear magnetic resonance assignment of the imino protons of lambda OR3 operator deoxyribonucleic acid

Using solid-phase phosphite triester methods, we have synthesized both strands of the phage lambda OR3 DNA sequence, reannealed them, and studied the native operator duplex by high-resolution NMR at 500 MHz. At 7 degrees C the imino protons of the two terminal base pairs at each end have disappeared from the spectrum by exchange broadening. The 13 detectable imino resonances have been assigned to their respective base pairs in the duplex by using sequential nearest-neighbor NOE connectivity methods described previously. In cases where two imino protons overlap in the spectrum, spin diffusion was used to drive the cross-saturation further afield in order to produce second-order next-nearest-neighbor effects. The results show that the imino connectivity method can be used to unambiguously assign the imino proton spectrum of operator DNAs containing one to two full turns of the helix.     

Analysis of the 1H-NMR spectra of ferrichrome peptides. I. The non-amide protons

The thus far unexplored aliphatic region of the proton magnetic resonance spectra of ferrichrome peptides was investigated at 360 MHz. Six isomorphic diamagnetic analogues of the ferric cyclohexapeptide differing in the coordinated cation (AL³⁺ or Ga³⁺) and the amino acid composition were studied in d6 -DMSO solution. By use of a novel resolution enhancement technique which applies a sinusoidal half-wave window to the free induction decay combined with multiplication by an increasing exponential, the proton chemical shifts and spin-spin couplings were accurately measured. Homonuclear decoupling combined with Fourier difference spectroscopy was used to selectively extract resonances out of crowded spectral regions. The spectra revealed unique features of fine structure in the proton resonance lines. Thus, the conformation-dependent geminal coupling constants of glycyl α-protons were found to be constant throughout the suite of analogue peptides. A similar invariance was observed for the vicinal coupling constants between α-, β-, γ-, and δ-protons in the ornithyl side chains. Comparison of the proton spin–spin coupling constants with the crstallographic dihedral angles led to a unique stereochemical assignment of the side-chain resonances. The combined data sets of x-ray atomic coordinates and ¹H-nmr spin-spin coupling parameters have been used to calibrate the coefficients for a Karplus curve related to the torsional x angles in amino acid side chains: Structurial information was also obtained for the seryl residues, where the multiplet structures of the OH resonances indicate preferred spatial arrangements of the side chains.     

C1 and cro repressors of lambda phages. I. Construction of vectors for expression of cro repressor of bacteriophage lambda imm434

Eight derivatives of recombinant plasmid pBRcro434, that consists of pBR322 and fragment of immunity region of phage lambda imm434 have been constructed and characterised. These derivatives contain the deletions in the region adjacent to OR3 operator and in the structural gene of cro-repressor of lambda imm434. The deletions have been produced by the treatment of pBRcro434 with exonuclease III of Escherichia coli and S1 nuclease of Aspergillus orizae and precisely mapped. The unique EcoRI-restriction sites have been reconstructed with the aim of using this deletion plasmids as a vectors for cloning.     

1H-NMR Of U-G-A and U-G-A-A in D2O:: Assignment of nonexchangeable protons and analysis of solution conformation

The ribonucleotide oligomers U-G-A and U-G-A-A have been synthesized enzymatically. These oligomers are cognates of the U³³-Gm³⁴-A³⁵-A³⁶ sequence found in the anticodon loop of t-RNA^phe. The ¹H-NMR chemical shifts of the base and ribose HI' protons as well as the couplings. J_1'–2', of the ribose protons have been examined as a function of temperature. Assignments for these resonances have been completed, and used in the analysis of solution conformation for these oligomers. The results are consistent with the A-RNA structure and suggest the absence of alternative ordered solution structures.     

Suppression of polarity of insertion mutations in the gal operon and N mutations in bacteriophage lambda.

Bacterial mutations (psuA and psu) known for their ability to suppress the polarity on nonsense mutations are shown to suppress the polarity of certain insertion mutations in the gal operon. The short insertion, IS1 (800 nucleotide pairs), is about 15 to 50% suppressed, whereas longer insertions, IS2 (1,400 nucleotide pairs), and IS3 (1,200 nucleotide pairs), are not. Some of the polarity suppressor mutations (psu-1, psu-2, and psu-3) are at least partially permissive for N-gene mutations (N7 and N53) of bacteriophage lambda, suggesting a relationship between natural and mutational polar signals. That this relationship may be complex is indicated by the fact that other suppressor mutations, effective in suppressing nonsense or insertion polarity, fail entirely to permit the growth of lambda N mutants.     

Long-range imino proton-13C J-couplings and the through-bond correlation of imino and non-exchangeable protons in unlabeled DNA

Thanks to rather large (5–9 Hz) long-range imino proton-¹³C J-couplings, heteronuclear correlation experiments in H₂O provide unambiguous assignment of imino protons by intranucleotide through-bond connectivities to guanosine H8 and thymidine CH₃ protons, or sequence-specific assignment of non-exchangeable protons when the imino protons are identified independently. This method is demonstrated in the Dickerson dodecamer [d(CGCGAATTCGCG)]₂ and in a human telomeric fragment of 22 nucleotides.     

Exclusion of bacteriophage T1 by bacteriophage lambda. I. Early exclusion requires lambda N gene product and host factors involved in N gene expression.

Two modes of exclusion of T1 by lambda are distinguished. "Early" exclusion depends on gene N, but not on gene Q. It is at least partially ineffective against T1am23. "Late" exclusion depends on gene Q and effects T1am23 as well as T1+. Early exclusion is a direct effect of N gene product, rather than N gene being required for the expression of some other lambda gene. Three host mutations, groN, nusA, and nusB, known to interfere with lambda replication by affecting N gene expression, also interfere with the ability of lambda to exclude T1.     

Translational regulatory signals within the coding region of the bacteriophage lambda cIII gene.

Six independent mutations which enhance the lysogenic response were analyzed. The mutations cause single-base substitutions at three sites within the cIII coding sequence, one of which does not change the amino acid code. The mutations allow for elevated translation of the cIII gene, possibly via changes in the mRNA secondary structure.