Richard H. Ebright

Dynamically driven protein allostery

Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.

Promoter structure, promoter recognition, and transcription activation in prokaryotes.

Steve Busby’ and Richard H. Ebrightt ‘School of Biochemistry University of Birmingham Birmingham 615 2lT England TDepartment of Chemistry and Waksman Institute Rutgers University New Brunswick, New Jersey 08855 Escherichia coli RNA polymerase holoenzyme (RNAP) has been the object of intense study since its discovery. RNAP consists of core enzyme, with subunit composition

Transcription activation at Class I CAP‐dependent promoters

Transcription activation at Class II CAP‐dependent promoters provides a paradigm for understanding how a single activator molecule can make multiple interactions with the transcription machinery, with each interaction being responsible for a specific mechanistic consequence. At Class II CAP‐dependent promoters, the DNA target site for CAP is centred near position −42, overlapping and replacing the −35 determinant for binding of RNA polymerase (RNAP). Transcription activation requires two distinct mechanistic components. The first component is ‘anti‐inhibition,’ overcoming an inhibitory effect of the RNAP α subunit C‐terminal domain (αCTD). This component involves direct contact between amino acids 156–164 (activating region 1) of the upstream subunit of the CAP dimer and a target in αCTD. The second component is ‘direct activation’, facilitating isomerization of the RNAP–promoter closed complex to the transcriptionally competent open complex. This component involves direct contact between amino acids 19, 21 and 101 (activating region 2) of the downstream subunit of the CAP dimer and a target in the RNAP α subunit N‐terminal domain (αNTD).

Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism.

Using fluorescence resonance energy transfer to monitor distances within single molecules of abortively initiating transcription initiation complexes, we show that initial transcription proceeds through a “scrunching” mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA into itself and past its active center. We show further that putative alternative mechanisms for RNAP active-center translocation in initial transcription, involving “transient excursions” of RNAP relative to DNA or “inchworming” of RNAP relative to DNA, do not occur. The results support a model in which a stressed intermediate, with DNA-unwinding stress and DNA-compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of interactions between RNAP and promoter DNA and between RNAP and initiation factors during promoter escape.

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA “scrunching”—in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself—and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.

Transcription Activation at Class II CAP-Dependent Promoters: Two Interactions between CAP and RNA Polymerase

At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase. We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain. We further show that the two interactions affect different steps in transcription initiation. Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence.

Domain organization of RNA polymerase α subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding

Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2). Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet). Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity. Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins.

Structural basis of transcription initiation.

The bacterial transcription initiation complex preorganizes promoter DNA for nucleotide binding and RNA synthesis. To transcribe a gene, RNA polymerase (RNAP) must unwind the promoter DNA to form a “transcription bubble” and the RNAP-promoter open complex (RPo). The activity of RPo is critical for the regulation of gene expression. Zhang et al. (p. 1076, published online 18 October) describe crystal structures of bacterial RPo, together with the transcription initiation factor σ, from Thermus thermophilus, variously in complex with promoter DNA and an RNA primer. RNAP and σ recognize the promoter through sequence-specific contacts with transcription-bubble, nontemplate-strand DNA. Critical interactions occur through the unstacking of DNA bases and their insertion into pockets on the surfaces of the two proteins, allowing direct sensing of the DNA sequence. During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, σA, and a promoter DNA fragment corresponding to the transcription bubble and downstream double-stranded DNA of the RNAP-promoter open complex. The structures show that σ recognizes the –10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases and that RNAP recognizes the –4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between σ and template-strand single-stranded DNA (ssDNA) preorganize template-strand ssDNA to engage the RNAP active center.

Structural Organization of the RNA Polymerase-Promoter Open Complex

We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.

Translocation of σ70 with RNA Polymerase during Transcription

Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.