Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described.

We have studied the properties and structures of a series of Escherichia coli RNA polymerase ternary complexes formed during the initial steps of RNA chain initiation and elongation. Five different templates were used that contained the bacteriophage T7 A1 promoter or the E. coli Tac or the lac UV5 promoter, as well as variant templates with alterations in the initial transcribed regions. The majority of ternary complexes bearing short transcripts (from two to nine nucleotides) are highly unstable and cannot be easily studied. This includes transcripts from the phage T7 A1 promoter, for which the stability of complexes bearing transcripts as short as four nucleotides has previously been postulated. However, with one Tac promoter template, RNA polymerase forms ternary complexes with transcripts as short as five nucleotides that are stable enough for biochemical study. We describe several approaches to identifying and isolating such stable complexes and show that stringent criteria are needed in carrying out such experiments if the results are to be meaningful. Deoxyribonuclease I (DNase I) footprinting has been used to probe the general structure of the stable ternary complexes formed as the polymerase begins transcription and moves away from the start site. The enzyme undergoes a sequence of structural changes during initiation and transition to an elongating complex. Complexes with five to eight nucleotide transcripts, designated initial transcribing complexes (ITC), have identical footprints; they all retain the sigma factor and have a slightly extended DNase I footprint (-57 to +24) as compared to the open promoter complex (-57 to +20). ITC complexes all show a region of marked DNase I hypersensitivity in the -25 region that may reflect bending or distortion of the DNA template. Complexes with 10 or 11 nucleotide transcripts, designated initial elongating complexes (IEC), have lost the sigma factor and have a slightly reduced and shifted DNase I footprint (-32 to +30). However, these IEC have not yet achieved the much smaller footprint (approximately 30 bp) reported as characteristic of elongating ternary complexes bearing longer RNA chains. During the initial phase of transcription, the RNA polymerase does not move monotonically along the DNA template as RNA chains are extended, but instead, the upstream and downstream contacts remain more or less fixed as the nascent transcript is elongated up to about eight nucleotides in length. Only after incorporation of 10 nucleotides is there significant movement of the enzyme away from the promoter region and a commitment to elongation.

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A mutant of Saceharomyces cereviswae with the genotype mnnl mnn2 mnn9 gisl synthesizes mannoproteins with oligosaccharides having the composition Glc3Man1OGlc-NAc2-owing to the mnn9 defect, which prevents synthesis of the outer chain, the mnni defect, which prevents branching of the core, and the gIsI mutation, which prevents deglucosylation of the resultant glycoprotein as a consequence of a defective glucosidase-I [Tsai, P.-K., Ballou, L., Esmon, B., Schekman, R. & Ballou, C. E. (1984) Proc. Natl. Acad. Sci. USA 81, [6340][6341][6342][6343]. (The mnn2 defect is not expressed in presence of the mnn9 mutation.) This strain spontaneously forms new colonies in which gisi is suppressed owing to a defect in synthesis of dolichol phosphoglucose, the glucosylation substrate. The new mutant, designated mnnl mnn2 mnn9 gisl dpgl, synthesizes and secretes invertase (EC 3.2.1.26) that has a higher mobility on native gel electrophoresis than that made by the parent strain, the consequence of a reduction in both the size and the number of carbohydrate chains. The mannoprotein chains have the mnnl mnn9 structure (Man1OGlc-NAc2-), and the invertase is resolved by gel electrophoresis in sodium dodecyl sulfate into two major and two minor bands that represent homologs with about 4-7 carbohydrate units, in contrast to about 8-11 chains in the parent strain. Thus, the inability to glucosylate the lipid-linked precursor reduces the efficiency of glycosylation of the protein chains. The genetic defect is in synthesis of the glucose donor dolichol phosphoglucose, but the mutation is nonallelic with the reported aigS-I mutation, which has a similar phenotype [Runge, K. W., Huffaker, T. C. & Robbins, P. W. (1984) Several studies have suggested that the glucose units on the dolichol-linked oligosaccharide precursor facilitate the transfer of the carbohydrate part to asparagine in polypeptide chains (1-3). On the other hand, a mutant of Saccharomyces cerevisiae that is unable to remove the glucose units from the oligosaccharide chains on glycosylated proteins (4), owing to a defect (glsl) in glucosidase-I (5), appears to act normally in the subsequent processing steps.We have taken advantage of the simplified carbohydrate chains made in the mnnl mnn2 mnn9 mutant of S. cerevisiae (6) to demonstrate the presence of the glucose units on invertase (EC 3.2.1.26) made in the mnnl mnn2 mnn9 gisl strain (7). The secreted invertases from both of these strains migrate on a nondenaturing gel as relatively sharp bands, with that from the strain with the gisi mutation traveling slightly slower than that from the mnnl mnn2 mnn9 strain owing to the extra hexose units. We have now observed that spontaneous mutations occur in the mnni mnn2 mnn9 gIsI strain that lead to colonies with a slightly altered morphology, the cells of which secrete invertase that has a higher electrophoretic mobility than the mnnl mnn2 mnn9 strain, even though the mannoproteins made in this new mutant have carbohydrate chains that lack the glucose units and are identical...