During T4 infection, transcriptional control is accomplished by phage- encoded factors that alter the specificity of the host RNA polymerase. Using an in vitro transcription system composed of T4-modified host RNA polymerase and the T4 transcriptional activator MotA, we are investigating the action of MotA at the T4 middle promoter PuvsX. We have found that transcription from PuvsX in vitro is sensitive to the addition of the polyanion heparin before but not after the addition of MotA. This result suggests that MotA is needed to form an open complex at PuvsX (the complex in which polymerase has partially unwound the promoter) since open complexes are known to be heparin resistant. We have partially purified and characterized a mutant MotA protein, MotA21 in which the first 8 amino acids of MotA have been substituted with 11 different amino acids. Although MotA21 binds to the PuvsX promoter nearly as well as MotA, MotA21 does not support transcription from PuvsX in vitro. These results are consistent with the MotA21 mutation affecting a domain of MotA that is involved in transcriptional activation rather than DNA binding. The T4 gene SegA encodes a protein that shares regions of similarity with a family of group I intron-encoded endonucleases present in fungi and phage. These proteins are required for the movement ('homing') of the intron DNA into its intronless gene, cutting at or near the site of intron insertion. Like these endonucleases, SegA is a Mg++-dependent DNA endonuclease which cuts with some sequence specificity, but it does not appear to be associated with an intron. We have used primer extension analyses to determine the exact positions of cutting by SegA at three preferred sites. While these three sites share some sequence elements, a consensus sequence cannot be deduced. This suggests that either SegA does not require a set of invariant bases or it recognizes some feature of the DNA other than sequence alone. From a screening of the genomes of 30 T- even like phage, we find that the SegA gene appears to be missing in all these genomes, consistent with the idea that the SegA gene was or is mobile.