William Whalen

An antitermination protein engages the elongating transcription apparatus at a promoter-proximal recognition site

As a transcriptional activator, the N protein of phage lambda acts to suppress transcription termination by recognizing a promoter-proximal site, nut, which is separated from the terminators by thousands of base pairs. We demonstrate here that N interacts with the elongating RNA polymerase in transit through the boxB domain of nut. This interaction leads to the stable association of N as an integral component of the transcription apparatus. During subsequent elongation, N translocates along with polymerase through several defined terminators positioned beyond nut. Therefore, by being an operon-specific subunit of the transcription apparatus, N presumably prevents the interaction of polymerase with termination signals.

Components of multiprotein-RNA complex that controls transcription elongation in Escherichia coli phage lambda

Publisher Summary This chapter discusses the components of multiprotein-RNA complex that controls transcription elongation in Escherichia Coli phage λ. Studies of bacteriophage A led to the discovery of several basic mechanisms of transcriptional regulation. One of these is transcriptional antitermination, the process in which genes whose transcription is otherwise blocked by premature termination are expressed through termination suppression. In λ and related phages, genome-specific antiterminators convert RNA polymerase (RNAP) into a termination-resistant form during early phases of transcription elongation. The chapter describes the current understanding of how one such antiterminator, the λ N gene product, works. The chapter reviews the methods of overproduction, isolation, and assay of the N protein as well as several accessory factors that modulate transcription elongation in Escherichia coli in the form of a multiprotein-RNA complex. The availability of N and Nus factors in large scale should now make it possible not only to attempt to determine the structures of the individual protein components, and the various RNA-protein and protein-protein complexes by biophysical methods, but also to examine these interactions by conventional biochemical methods.

IMMUNOPRINTING: A TECHNIQUE USED TO STUDY DYNAMIC PROTEIN-NUCLEIC ACID INTERACTIONS WITHIN TRANSCRIPTION ELONGATION COMPLEX

Publisher Summary This chapter discusses the immunoprinting technique to study dynamic protein-nucleic acid interactions within transcription elongation complex. Immunoprinting is used to locate the sites within the phage λ genome where the phage N gene product specifically captures Escherichia coli RNA polymerase (RNAP). It provided evidence that an antiterminator such as N can become an integral subunit of the transcription machinery once it captures RNAP at a genetically defined site. The chapter describes the basic strategies of the immunoprinting method, its application to the N system, and the implication of the findings on the mechanism of N action. The N antiterminator suppresses both Rho-dependent and Rho-independent terminators on the λ genome that are located within the two early operons at many intergenic sites. Two simple models can explain how the nut site acts at a distance to mediate N antitermination. Both models invoke an interaction between N and nut, as well as an interaction between N and polymerase.