Abstract: Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences within a complex and heterogeneous chromatin environment to assemble transcriptional machinery at specific genomic loci. The mechanisms by which TFs bind to their target sequences on dynamic chromatin to regulate gene expression remains elusive. Single-molecule tracking (SMT) has emerged as a powerful approach to explore chromatin and transcription factor dynamics and their interaction in living cells. We used single-molecule tracking to directly measure the interaction dynamics of a broad spectrum of transcription factors in live cells. We found that TFs follow power-law distributed binding times, suggesting that the prevalent model of specific and non-specific TF/chromatin interactions is incomplete. Using machine-learning based analysis of single molecule mobility, we show that chromatin displays two distinct low-mobility states. Our experimental observations are consistent with a minimal active copolymer model for interphase chromosomes. Remarkably, we find that a diverse set of transcription factors, co-regulators and remodelers also exhibit two distinct low-mobility states. Mutational analysis reveals that interactions with chromatin in the lowest-mobility state require an intact DNA binding domain and oligomerization domains. Our observations further suggest that intrinsically disordered regions which are ubiquitous in many transcription factors, are a key determinant of the second low mobility state. Together, our results elucidate how transcription factor and chromatin mobility regulates transcriptional activation in mammalian cells.