Ribosome reactivates transcription by physically pushing RNA polymerase out of transcription arrest.

In bacteria, the first two steps of gene expression-transcription and translation-are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription-translation coupling mitigates these conflicts is unknown.

Phosphorylation decelerates conformational dynamics in bacterial translation elongation factors.

Bacterial protein synthesis is intricately connected to metabolic rate. One of the ways in which bacteria respond to environmental stress is through posttranslational modifications of translation factors. Translation elongation factor Tu (EF-Tu) is methylated and phosphorylated in response to nutrient starvation upon entering stationary phase, and its phosphorylation is a crucial step in the pathway toward sporulation.

Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate.

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP.

Methodology for the analysis of transcription and translation in transcription-coupled-to-translation systems in vitro.

The various properties of RNA polymerase (RNAP) complexes with nucleic acids during different stages of transcription involve various types of regulation and different cross-talk with other cellular entities and with fellow RNAP molecules. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of outcomes of gene expression, whereas the study of the physical interaction of the ribosome and the RNAP remains obscure partly due to the lack of a system that allows such observations. In this article we will describe the methodology needed to set up a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing investigation of the interactions between the two machineries at colliding and non-colliding distances.

Methods for the assembly and analysis of in vitro transcription-coupled-to-translation systems.

RNA polymerase is a complex machinery, which is further embedded in interactions with other cellular components that interplay with either the transcribed DNA (DNA polymerases, topoisomerases, etc.) or the nascent RNA (RNA processing enzymes, ribosomes, etc.).

The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu.

Fic proteins are ubiquitous in all of the domains of life and have critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc-phd toxin-antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to having AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering EF-Tu unable to bind aminoacylated tRNAs.

Molecular mechanism of bacterial persistence by HipA.

HipA of Escherichia coli is a eukaryote-like serine-threonine kinase that inhibits cell growth and induces persistence (multidrug tolerance). Previously, it was proposed that HipA inhibits cell growth by the phosphorylation of the essential translation factor EF-Tu. Here, we provide evidence that EF-Tu is not a target of HipA.

In vitro experimental system for analysis of transcription-translation coupling.

Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription-translation, both machineries mutually affect each others' functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system.

Extensive functional overlap between sigma factors in Escherichia coli.

Bacterial core RNA polymerase (RNAP) must associate with a sigma factor to recognize promoter sequences. Escherichia coli encodes seven sigma factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E.

Transcription regulatory elements are punctuation marks for DNA replication.

Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven.