Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii.

Understanding the molecular basis of regulated nitrogen (N) fixation is essential for engineering N-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability.

Initiation of Translation in Bacteria and Chloroplasts.

Relative rates of protein synthesis in bacteria generally depend on the number of copies of a messenger RNA (mRNA) and the efficiency of their loading with ribosomes. Translation initiation involves the multi-stage assembly of the ribosome on the mRNA to begin protein synthesis. In bacteria, the small ribosomal subunit (30S) and mRNA form a complex that can be supported by RNA-protein and RNA-RNA interactions and is extensively modulated by mRNA folding.

A disease resistance protein triggers oligomerization of its NLR helper into a hexameric resistosome to mediate innate immunity.

NRCs are essential helper NLR (nucleotide-binding domain and leucine-rich repeat) proteins that execute immune responses triggered by sensor NLRs. The resting state of NbNRC2 was recently shown to be a homodimer, but the sensor-activated state remains unclear. Using cryo-EM, we determined the structure of sensor-activated NbNRC2, which forms a hexameric inflammasome-like resistosome.

Structure of the plant plastid-encoded RNA polymerase.

Chloroplast genes encoding photosynthesis-associated proteins are predominantly transcribed by the plastid-encoded RNA polymerase (PEP). PEP is a multi-subunit complex composed of plastid-encoded subunits similar to bacterial RNA polymerases (RNAPs) stably bound to a set of nuclear-encoded PEP-associated proteins (PAPs). PAPs are essential to PEP activity and chloroplast biogenesis, but their roles are poorly defined.

Structural insights into RNA-mediated transcription regulation in bacteria.

RNA can regulate its own synthesis without auxiliary proteins. For example, U-rich RNA sequences signal RNA polymerase (RNAP) to pause transcription and are required for transcript release at intrinsic terminators in all kingdoms of life. In contrast, the regulatory RNA putL suppresses pausing and termination in cis.

Macromolecular assemblies supporting transcription-translation coupling.

Coordination between the molecular machineries that synthesize and decode prokaryotic mRNAs is an important layer of gene expression control known as transcription-translation coupling. While it has long been known that translation can regulate transcription and vice-versa, recent structural and biochemical work has shed light on the underlying mechanistic basis. Complexes of RNA polymerase linked to a trailing ribosome (expressomes) have been structurally characterized in a variety of states at near-atomic resolution, and also directly visualized in cells.

Structural basis of transcription-translation coupling and collision in bacteria.

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The lead ribosome potentially contacts RNA polymerase (RNAP) and forms a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood.

Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity .

Tristetraprolin (TTP) is an anti-inflammatory protein that modulates the stability of certain cytokine/chemokine mRNAs. After initial high-affinity binding to AU-rich elements in 3' untranslated regions of target mRNAs, mediated through its tandem zinc finger (TZF) domain, TTP promotes the deadenylation and ultimate decay of target transcripts. These transcripts and their encoded proteins accumulate abnormally in TTP knockout (KO) mice, leading to a severe inflammatory syndrome.

RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not.

The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity by interacting with both Ccr4-Not and target mRNAs, but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs).

mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases.

Translation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases-Ccr4 and Caf1/Pop2-that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes.

Analysis of mRNA deadenylation by multi-protein complexes.

Poly(A) tails are found at the 3' end of almost every eukaryotic mRNA and are important for the stability of mRNAs and their translation into proteins. Thus, removal of the poly(A) tail, a process called deadenylation, is critical for regulation of gene expression. Most deadenylation enzymes are components of large multi-protein complexes.

Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1.

Ccr4-Not is a conserved protein complex that shortens the 3' poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates.

Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite's Chloroquine Resistance Transporter.

Mutations in the Plasmodium falciparum 'chloroquine resistance transporter' (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite's digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite's hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs.

The yeast transcription elongation factor Spt4/5 is a sequence-specific RNA binding protein.

The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats.

The identification and structure of an N-terminal PR domain show that FOG1 is a member of the PRDM family of proteins.

FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecognized domain in FOG1: a PR (PRD-BF1 and RIZ) domain that is distantly related in sequence to the SET domains that are found in many histone methyltransferases.

Dissecting the mechanism of abscisic acid-induced dynamic microtubule reorientation using live cell imaging.

Abscisic acid (ABA) is involved in plant development and responses to environmental stress including the formation of longitudinal microtubule arrays in elongating cells, although the underlying mechanism for this is unknown. We explored ABA-induced microtubule reorientation in leek (Allium porrum L.) leaf epidermal cells transiently expressing a GFP-MBD microtubule reporter.