Amanda S. Altieri

Allosteric Activation of E2-RING Finger-Mediated Ubiquitylation by a Structurally Defined Specific E2-Binding Region of gp78

The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.

Breaking symmetry in the structure determination of (large) symmetric protein dimers

We demonstrate a novel methodology to disrupt the symmetry in the NMR spectra of homodimers. A paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on only one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. This methodology can be widely used for solution structure determination of symmetric dimers.

The structure of the transcriptional antiterminator NusB from Escherichia coli

We have determined the solution structure of NusB, a transcription antitermination protein from Escherichia coli. The structure reveals a novel, all alpha-helical protein fold. NusB mutations that cause a loss of function (NusB5) or alter specificity for RNA targets (NusB101) are localized to surface residues and likely affect RNA-protein or protein-protein interactions. Residues that are highly conserved among homologs stabilize the protein core. The solution structure of E. coli NusB presented here resembles that of Mycobacterium tuberculosis NusB determined by X-ray diffraction, but differs substantially from a solution structure of E. coli NusB reported earlier.

Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex.

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.

Structural Biophysics of the NusB:NusE Antitermination Complex

In prokaryotic transcription regulation, several host factors form a complex with RNA polymerase and the nascent mRNA. As part of a process known as antitermination, two of these host factors, NusB and NusE, bind to form a heterodimer, which interacts with a specific boxA site on the RNA. The NusB/NusE/boxA RNA ternary complex interacts with the RNA polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. The NusB protein also binds boxA RNA individually and retains all specificity for boxA. However, NusE increases the affinity of RNA to NusB in the ternary complex, which contributes to efficient antitermination. To understand the molecular mechanism of the process, we have determined the structure of NusB from the thermophilic bacterium Aquifex aeolicus and studied the interaction of NusB and NusE. We characterize this binding interaction using NMR, isothermal titration calorimetry, gel filtration, and analytical ultracentrifugation. The binding site of NusE on NusB was determined using NMR chemical shift perturbation studies. We have also determined the NusE binding site in the ternary Escherichia coli NusB/NusE/boxA RNA complex and show that it is very similar to that in the NusB/NusE complex. There is one loop of residues (from 113 to 118 in NusB) affected by NusE binding in the ternary complex but not in the binary complex. This difference may be correlated to an increase in binding affinity of RNA for the NusB/NusE complex.

Sequential assignments and secondary structure of the RNA-binding transcriptional regulator NusB

The NusB protein is involved in transcriptional regulation in bacteriophage λ. NusB binds to the RNA form of the nut site and along with N, NusA, NusE and NusG, stabilizes the RNA polymerase transcription complex and allows stable, persistent antitermination. NusB contains a 10 residue Arg‐rich RNA‐binding motif (ARM) at the N‐terminus but is not sequentially homologous to any other proteins. In contrast to other known ARM‐containing proteins, NusB forms a stable structure in solution in the absence of RNA. NMR spectroscopy was used to determine that NusB contains six α‐helices: R10–Q21, I27–F34, V45–L65, Q79–S93, Y100–F114 and D118–L127. The structure of NusB makes it a member of a newly emerging class of α‐helical RNA‐binding proteins.

Crystal structure of a plectonemic RNA supercoil

Genome packaging is an essential housekeeping process in virtually all organisms for proper storage and maintenance of genetic information. Although the extent and mechanisms of packaging vary, the process involves the formation of nucleic-acid superstructures. Crystal structures of DNA coiled coils indicate that their geometries can vary according to sequence and/or the presence of stabilizers such as proteins or small molecules. However, such superstructures have not been revealed for RNA. Here we report the crystal structure of an RNA supercoil, which displays one level higher molecular organization than previously reported structures of DNA coiled coils. In the presence of an RNA-binding protein, two interlocking RNA coiled coils of double-stranded RNA, a ‘coil of coiled coils’, form a plectonemic supercoil. Molecular dynamics simulations suggest that protein-RNA interaction is required for the stability of the supercoiled RNA. This study provides structural insight into higher-order packaging mechanisms of nucleic acids.