Table 15.1 Characteristics of Popular Prokaryotic Promoters Regulation/Inducer Promoter (Concentration LacUv5 Lactose operon lacI/IPTG Strong (0.1-1mM) Ip trpR 3- Strong beta-indoleacrylic Hybrid of -35 lacI/IPTG Strong Trp and -10 (0.1-1mM) P Phage lambda ambda cl Strong Phage T5 lacl (2 operators)/ Strong IPTG(0.1-1 mM) Arabinose Arabinose Ara badlarabinose (1m-10mM) T7 phage RNA lacI/m ery strong (0.1-1mM) A transcriptional promoter. A ribosome binding site. a translation initiation site A selective marker(e.g, antibiotic resistance) An origin of replication In general, things that affect these can affect the level of protein xpression. At a minimum, transcription promoters in E. coli consist of two dna hexamers located -35 and -10 relative to the transcriptional start site. Together these elements mediate binding of the about 500kDa multimeric complex of Rna polymerase e. Suppliers of vectors for expression have selected highly active, inducible promoter sequences, and there is usually little need to be concerned until a problem is encountered in expression. A list of the commonly used promoters and their regulation is shown in Table 15.1 Is a Stronger Promoter Always Desirable? A strong promoter may not be best for all situations Over- production of RNa may saturate translation machinery, and maximizing RNa synthesis may not be desirable or necessary. A weaker promoter may actually give higher steady-state levels of soluble, intact protein than one that is rapidly induced E coli Expression Systems• A transcriptional promoter. • A ribosome binding site. • A translation initiation site. • A selective marker (e.g., antibiotic resistance). • An origin of replication. In general, things that affect these can affect the level of protein expression. At a minimum, transcription promoters in E. coli consist of two DNA hexamers located -35 and -10 relative to the transcriptional start site.Together these elements mediate binding of the about 500kDa multimeric complex of RNA polymerase. Suppliers of vectors for expression have selected highly active, and inducible promoter sequences, and there is usually little need to be concerned until a problem is encountered in expression. A list of the commonly used promoters and their regulation is shown in Table 15.1. Is a Stronger Promoter Always Desirable? A strong promoter may not be best for all situations. Over￾production of RNA may saturate translation machinery, and maximizing RNA synthesis may not be desirable or necessary. A weaker promoter may actually give higher steady-state levels of soluble, intact protein than one that is rapidly induced. E. coli Expression Systems 463 Table 15.1 Characteristics of Popular Prokaryotic Promoters Regulation/Inducer Promoter (Concentration) Strength LacUV5 Lactose operon lacI/IPTG Strong (0.1–1 mM) Trp Tryptophan trpR 3- Strong operon beta-indoleacrylic acid Tac Hybrid of -35 lacI/IPTG Strong Trp and -10 (0.1–1 mM) lac promoter PL Phage lambda Lambda cI Strong repressor/heat Phage T5 T5 phage lacI (2 operators)/ Strong IPTG (0.1–1mM) Arabinose Arabinose AraBAD/arabinose Variable operon (1mm–10 mM) T7 T7 phage RNA lacI/IPTG Very strong polymerase (0.1–1 mM)