The power of the small: the underestimated role of small proteins in bacterial and archaeal physiology.

Small proteins encoded by small open-reading frames (sORFs) (≤70 aa) were overlooked for decades due to methodological reasons and are thus often missing in genome annotations. Novel detection methods such as ribosome profiling (Ribo-Seq) and mass spectrometry optimized for small proteins (peptidomics) have opened up a new field of interest and several catalogs of small proteins in bacteria and archaea have been recently reported. Many translated sORFs have been discovered in genomic locations previously thought to be noncoding, such as 5' or 3' untranslated regions or well-studied regulatory small RNAs (sRNAs). Even within longer ORFs, additional functional sORFs have been detected. Today, only a small proportion is characterized, but those small proteins indicate important and diverse functions in cellular physiology. Here, we summarize recently characterized small proteins involved in microbial metabolism.

Microscale Thermophoresis to Study RNA-RNA Binding Affinity.

Evaluation of RNA-RNA binding is crucial for in vitro studying of molecular mechanisms, for example, the interaction of noncoding RNAs (ncRNAs) with their respective targets. In recent years, the method of microscale thermophoresis (MST) has been developed, which is based on the physical phenomenon of thermophoresis (Ludwig-Soret Effect), defined as the migration of a molecule in a solution in response to a macroscopic temperature gradient. The method enables the fast detection and characterization of biophysical interaction between molecules, with the fundamental advantage that only small amounts of target and ligand are required. Here, we describe the characterization of RNA-RNA binding affinity using the example of the sRNA41 from Methanosarcina mazei and its native target, the 5' UTR of mRNA-MM2089, the first gene of the operon encoding the acetyl-CoA decarboxylase/synthase complex.

High complexity of Glutamine synthetase regulation in Methanosarcina mazei: Small protein 26 interacts and enhances glutamine synthetase activity.

Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- and noncoding-predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull-down by affinity chromatography followed by MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1 ) with high affinity (app. KD  = 0.76 µm± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1 . Upon interaction with sP26, GlnA1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2-oxoglutarate (2-OG). Besides, strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (app. KD  = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1 .