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

MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering

Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk

Nus transcription elongation factors and RNase III modulate small ribosome subunit biogenesis in E scherichia coli

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96334/1/mmi12105.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96334/2/mmi12105-sup-0001-si.pd

Quick change: post-transcriptional regulation in Pseudomonas

Pseudomonas species have evolved dynamic and intricate regulatory networks to fine-tune gene expression, with complex regulation occurring at every stage in the processing of genetic information. This approach enables Pseudomonas to generate precise individual responses to the environment in order to improve their fitness and resource economy. The weak correlations we observe between RNA and protein abundance highlight the significant regulatory contribution of a series of intersecting post-transcriptional pathways, influencing mRNA stability, translational activity and ribosome function, to Pseudomonas environmental responses. This review examines our current understanding of three major post-transcriptional regulatory systems in Pseudomonas spp.; Gac/Rsm, Hfq and RimK, and presents an overview of new research frontiers, emerging genome-wide methodologies, and their potential for the study of global regulatory responses in Pseudomonas

Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes

Assembly of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. Here, we use three-color single molecule FRET to show how combinations of ribosomal proteins uS4, uS17 and bS20 in the 16S 5' domain enable the recruitment of protein bS16, the next protein to join the complex. Analysis of real-time bS16 binding events shows that bS16 binds both native and non-native forms of the rRNA. The native rRNA conformation is increasingly favored after bS16 binds, explaining how bS16 drives later steps of 30S assembly. Chemical footprinting and molecular dynamics simulations show that each ribosomal protein switches the 16S conformation and dampens fluctuations at the interface between rRNA subdomains where bS16 binds. The results suggest that specific protein-induced changes in the rRNA dynamics underlie the hierarchy of 30S assembly and simplify the search for the native ribosome structure

Origin of a folded repeat protein from an intrinsically disordered ancestor.

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2-5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin

Recent Acceleration of Plastid Sequence and Structural Evolution Coincides with Extreme Mitochondrial Divergence in the Angiosperm Genus Silene

The angiosperm genus Silene exhibits some of the most extreme and rapid divergence ever identified in mitochondrial genome architecture and nucleotide substitution rates. These patterns have been considered mitochondrial specific based on the absence of correlated changes in the small number of available nuclear and plastid gene sequences. To better assess the relationship between mitochondrial and plastid evolution, we sequenced the plastid genomes from four Silene species with fully sequenced mitochondrial genomes. We found that two species with fast-evolving mitochondrial genomes, S. noctiflora and S. conica, also exhibit accelerated rates of sequence and structural evolution in their plastid genomes. The nature of these changes, however, is markedly different from those in the mitochondrial genome. For example, in contrast to the mitochondrial pattern, which appears to be genome wide and mutationally driven, the plastid substitution rate accelerations are restricted to a subset of genes and preferentially affect nonsynonymous sites, indicating that altered selection pressures are acting on specific plastid-encoded functions in these species. Indeed, some plastid genes in S. noctiflora and S. conica show strong evidence of positive selection. In contrast, two species with more slowly evolving mitochondrial genomes, S. latifolia and S. vulgaris, have correspondingly low rates of nucleotide substitution in plastid genes as well as a plastid genome structure that has remained essentially unchanged since the origin of angiosperms. These results raise the possibility that common evolutionary forces could be shaping the extreme but distinct patterns of divergence in both organelle genomes within this genus