No abstract

To investigate the nature of the chemical determinants in DNA required for nonspecific binding and bending by proteins we have created a novel DNA in which inosine-5-methylcytosine and 2,6-diaminopurine-uracil base pairs are substituted for normal base pairs in a defined DNA sequence. This procedure completely switches the patterns of the base pair H bonding and attachment of exocyclic groups. We show that this DNA binds a histone octamer more tightly than normal DNA but, surprisingly, does not alter the orientation of the sequence on the surface of the protein. However, in general, the addition or removal of DNA exocyclic groups reduces or increases, respectively, the affinity for the histone octamer. The average incremental change in binding energy for a single exocyclic group is Ϸ40 J͞mol. The orientation of the DNA in core nucleosomes also is sensitive to the number and nature of the exocyclic groups present. Notably, substitutionwiththenaturallyoccurringcytosineanalogue,5-methylcytosine, shifts the preferred rotational position by 3 bp, whereas incorporating 2,6-diaminopurine shifts it 2 bp in the opposite direction. These manipulations potentially would alter the accessibility of a protein recognition sequence on the surface of the histone octamer. We propose that exocyclic groups impose steric constraints on protein-induced DNA wrapping and are also important in determining the orientation of DNA on a protein surface. In addition, we consider the implications of the selection of A-T and G-C base pairs in natural DNA.The local deformation of DNA by DNA-bending proteins can substantially exceed the normal conformational fluctuations of DNA free in solution. Although the interactions that determine the local sequence-dependent conformation of free DNA are relatively well understood (1-7), the nature of the chemical constraints that limit its further deformation have not been explored extensively. The nucleosome core particle in which the central 125 bp of DNA are wrapped in 1.6 superhelical turns about the histone octamer (8, 9) provides a good system to study this problem. In this particle both the major and minor grooves are compressed on the inside of the wrapped DNA and widened on the outside. A major determinant of the rotational placement of the DNA in the nucleosome is the periodic occurrence of short A͞T-rich sequences in helical phase and of short G͞C-rich sequences in the opposite phase. These short sequences favor local conformations with narrow and wide minor grooves, respectively, and thus together facilitate the tight wrapping of DNA (10-16).To investigate the nature of other chemical determinants that constrain the wrapping of DNA around the histone octamer we chose a prokaryotic DNA sequence derived from the Escherichia coli tyrT promoter. Previous studies have shown that the histone octamer occupies a preferred rotational position on this DNA (10). We modified this DNA by replacing the naturally occurring bases with analogues either lacking or containing additional exocyclic groups b...

After over a century of extensive research, hemoglobin has become the prototype of allosteric and cooperative proteins. Its molecular structure, known in great detail, has allowed the design of hundreds of site directed mutations, aimed at interfering with its function, and thus at testing our hypotheses on the molecular mechanisms of allostery. The wealth of information thus obtained is difficult to read except for specialists, not only because it makes use of many different technical approaches, but also because of its intrinsically patchy nature. Moreover, several researchers have tried to assign specific roles to segments of the polypeptide chains, rather than to single residues, and have tested their hypotheses by multiple point mutations or by complete replacement with the homologous segment from a different hemoglobin to produce chimeric macromolecules. This approach is in great need of a revision since putative functionally relevant segments partially overlap. This review briefly describes the structure and function of hemoglobin, and analyzes the effect of point mutations, multiple mutations and segment replacement, with special attention to possible biotechnological applications, ranging from pharmacology (Hb solutions as resuscitating fluids and sources of the protein found in hemoglobinopathies for biochemical studies) to bioreactors. Occasional reference is made to site directed mutants of myoglobin, whenever this helps clarifying perplexing results obtained on hemoglobin.

In addition to the well-known internal promoter elements of tRNA genes, 5' flanking sequences can also influence the efficiency of transcription by Saccharomyces cerevilsae extracts in vitro. A consensus sequence of yeast tRNA genes in the vicinity of the transcriptional start site can be derived. To determine whether the activity of this region can be attributed to particular sequence features we studied in vitro mutants of the start site region. We found that the start site can be shifted, but only to a limited extent, by moving the conserved sequence element. We found that both a pyrimidine-purine motif (with transcription initiating at the purine) and a small T:A base pair block upstream are important for efficient transcription in vitro. Thus the sequence surrounding the start site of transcription of the yeast tRNAL1u3 gene does play a role in determining transcription efficiency and fixing the precise site of initiation by RNA polymerase Ill.

XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3(1)21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution.