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Human serum albumin prepared by blood fractionation for clinical purposes was found to degrade when stored at or above 30°C. Mass spectrometry and N-terminal sequencing of the protein identified degradation corresponding to the loss of the first two residues, aspartic acid and alanine. The reaction was shown to be dependent upon temperature and the N-terminal a-amino group. In addition, comparison with serum albumins derived from other species showed that the instability of the N-terminus was specific to the human albumin sequence. An intact aspartyl-alanyl dipeptide, purified from degraded albumin solutio'ns, differed substantially from a synthetic dipeptide on amino acid analysis, N-terminal sequencing and NIMR. It is suggested that the released dipeptide may be cyclic, implying a novel cleavage mechanism.

Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region. Footprint analysis of transcriptionally competent complexes between E. coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed. In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected. Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA. However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence. With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced. In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone. We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation.

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