Identification of trans -acting factors that associate with ESS3a. RNA affinity columns were programmed withTRAP-tagged RNA transcripts of ESS3a or with a mutant of ESS3adesignated ESS3a 5-2. HeLa nuclear extract was passed over the columns and column eluates fractionated by SDS-PAGE and silver stained. ESS3a enriched bands were excised and sequenced by mass spectrometry. (A) Schematic representation of HIV-1 genome. Open boxes represent the open reading frames encoding the indicated viral proteins, shaded boxes indicate 5' and 3' LTRs. Also shown is a schematic of the TRAP-ESS vector. The construct contains a Streptavidin binding aptamer, S1 and two MS2 coat protein binding sites followed by the ESS3a sequence. The sequence of ESS3a is shown. The altered nucleotides of the mutant designated ESS3a 5-2 are indicated above the wild-type sequence. (B) Silver stain of proteins eluted from Streptavidin column with indicated RNA bait. Proteins were fractionated on 10% SDS-PAGE. The arrow indicates the excised band. Western blot against hnRNP E2; ESS or mutant thereof are as shown (C) Comparison of hnRNP E1 and hnRNP E2. Boxed is the sequence generated from the excised band analyzed by mass spectrometry.