A regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this two-domain protein refolds from an α-hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound β-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (α-hairpin) and active (β-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state (∼20% deuteron incorporation), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the β-folded state. Computationally-predicted ΔGs agree with these results by displaying similar anisotropic stability within the tip of the α-hairpin and on neighboring N-terminal domain residues. Remarkably, the β-folded state shows comparable stability to non-metamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins, and for devising targeted strategies to combat bacterial diseases. Significance Infections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gram-negative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression. However, the molecular basis of this unique conformational change is poorly understood. Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.

The coupling of transcription and translation is more than mere translation of an mRNA that is still being transcribed. The discovery of physical interactions between RNA polymerase and ribosomes has spurred renewed interest into this long-standing paradigm of bacterial molecular biology. Here, we provide a concise presentation of recent insights gained from super-resolution microscopy, biochemical, and structural work, including cryo-EM studies. Based on the presented data, we put forward a dynamic model for the interaction between RNA polymerase and ribosomes, in which the interactions are repeatedly formed and broken. Furthermore, we propose that long intervening nascent RNA will loop out and away during the forming the interactions between the RNA polymerase and ribosomes. By comparing the effect of the direct interactions between RNA polymerase and ribosomes with those that transcription factors NusG and RfaH mediate, we submit that two distinct modes of coupling exist: Factor-free and factor-mediated coupling. Finally, we provide a possible framework for transcription-translation coupling and elude to some open questions in the field.

Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. The Fur (ferric uptake regulation) protein is a negative regulator of the aerobactin operon and of several other siderophore-mediated, high-affinity iron transport systems in Escherichia coli. The purified Fur protein and a plasmid containing a lacZ fusion to the aerobactin operon were used in conjunction with an in vitro coupled transcription/translation system to demonstrate that the Fur protein requires Fe(II) or certain other divalent metals as a cofactor to negatively regulate expression of the aerobactin operon. In a second set of experiments, using a restriction site protection assay, Fur was shown to bind to and block the aerobactin promoter in a metal-dependent fashion. It is concluded that Fur acts as a classical negative repressor that, under in vivo conditions, uses ionic Fe(II) as a corepressor. Our results support the hypothesis [Williams, R.J.P. (1982) FEBS Lett. 140, 3-10] that prokaryotic cells may contain a standing pool of free or loosely bound Fe(II) that is capable of acting in a regulatory capacity.

Prokaryotic gene expression in vitro: transcription-translation coupled systems. Transcription-translation coupled systems have been developed to study prokaryotic gene expression. Several types of expression system have been described. The original system consists of a crude unfractionated Escherichia coli extract, which supports protein synthesis directed by a template DNA. Control of gene expression at the transcriptional stage has been studied using this unfractionated system. In this respect, two examples of particular interest, lactose and tryptophan operons, are described. Other systems are either partially reconstituted or highly defined, containing up to 30 purified factors necessary for transcription (RNA polymerase) and translation (aminoacyl-tRNA synthetases, initiation, elongation and release factors). Additional differences between the various systems relate to the analysis of the gene products. Whereas most methods involve analysis of the totally synthesized protein, a particular system implies the formation of only the N-terminal di- or tripeptide of the gene product.

591 Prokaryotic autotrophs, 139-40 Prokaryotic cells, 99, 589 replication, 218, 219-21 Prokaryotic vs … between transcription and translation in terms of their purpose and location. 7. Transcription and translation … phenomenon is known as coupled transcription-translation (Figure 1). 2. The genes of prokaryotic organisms do not

required to translate it is 4n-1. The rate of translation varies; it is significantly higher in prokaryotic cells (up to 17–21 amino acid residues per second) Translation is the process in biological cells in which proteins are produced using RNA molecules as templates. The generated protein is a sequence of amino acids determined by the sequence of nucleotides in the RNA. The nucleotides are considered three at a time. Each such triple results in the addition of one specific amino acid to the protein being generated. The matching from nucleotide triple Elongation depends on elongation factors. At the end of the initiation step, the mRNA is positioned so that the next codon can be translated during the elongation stage of protein synthesis. The initiator tRNA occupies the P site in the ribosome, and the A site is ready to receive an aminoacyl-tRNA. During chain elongation, each additional amino acid is added to the nascent polypeptide chain in a three-step microcycle. The steps in this microcycle are (1) positioning the correct aminoacyl-tRNA in the A site of the ribosome, which is brought into that site by eEF1, (2) forming the peptide bond, and (3) shifting the mRNA by one codon relative to the ribosome with the help of eEF2. Unlike bacteria, in which translation initiation occurs as soon as the 5' end of an mRNA is synthesized, in eukaryotes, such tight coupling between transcription and translation is not possible because transcription and translation are carried out in separate compartments of the cell (the nucleus and cytoplasm). Eukaryotic mRNA precursors must be processed in the nucleus (e.g., capping, polyadenylation, splicing) in ribosomes before they are exported to the cytoplasm for translation. Translation can also be affected by ribosomal pausing, which can trigger endonucleolytic attack of the tRNA, a process termed mRNA no-go decay. Ribosomal pausing also aids co-translational folding of the nascent polypeptide on the ribosome, and delays protein translation while it is encoding tRNA. This can trigger ribosomal frameshifting. The last tRNA validated by the small ribosomal subunit (accommodation) transfers the amino acid. It carries to the large ribosomal subunit which binds it to one of the preceding admitted tRNA (transpeptidation). The ribosome then moves to the next mRNA codon to continue the process (translocation), creating an amino acid chain. In bacterial translation, and archaeal translation, translation occurs in the cytosol, where the ribosome binds to the mRNA. In eukaryotes, translation can occur in the cytoplasm and also across the membrane of the endoplasmic reticulum through a process called co-translational translocation. In co-translational translocation, the entire…