adenosine-5--triphosphate-3--diphosphate and adenosine-3--diphosphate-5--diphosphate

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNu

Topics: Adenine Nucleotides; Allosteric Site; Bacillus subtilis; Bacterial Proteins; Deoxyribonucleotides; Escherichia coli; Gene Expression Regulation, Bacterial; Ligases; Protein Binding; Protein Conformation; Pyrophosphatases

Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5'triphosphate-3'diphosphate and guanosine 3', 5'-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5' and 3' moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic. Here, we use (p)ppGpp's adenine analogs [(p)ppApp] to investigate how the nucleobase contributes to determine its binding site and transcriptional regulation. We determined X-ray crystal structure of Escherichia coli RNAP-(p)ppApp complex, which shows the analogs bind near the active site and switch regions of RNAP. We have also explored the regulatory effects of (p)ppApp on transcription initiating from the well-studied E. coli rrnB P1 promoter to assess and compare properties of (p)ppApp with (p)ppGpp. We demonstrate that contrary to (p)ppGpp, (p)ppApp activates transcription at this promoter and DksA hinders this effect. Moreover, pppApp exerts a stronger effect than ppApp. We also show that when ppGpp and pppApp are present together, the outcome depends on which one of them was pre-incubated with RNAP first. This behavior suggests a surprising Yin-Yang like reciprocal plasticity of RNAP responses at a single promoter, occasioned simply by pre-exposure to one or the other nucleotide. Our observations underscore the importance of the (p)ppNpp's purine nucleobase for interactions with RNAP, which may lead to a better fundamental understanding of (p)ppGpp regulation of RNAP activity.

Topics: Adenine Nucleotides; Binding Sites; DNA-Directed RNA Polymerases; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Guanosine Pentaphosphate; Models, Molecular; Promoter Regions, Genetic; Structure-Activity Relationship; Transcriptional Activation

Topics: Adenine Nucleotides; Cell Differentiation; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cyanobacteria; Phosphorylation; Ribonucleotides