Abstract
Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of ...tRNAs, mRNAs, RNA half‐lives, proteins, and protein half‐lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium
Mycoplasma pneumoniae
(
Mpn
). We found that, although the initiation rate varied over 160‐fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100‐fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in
Mpn
.
Synopsis
image
Integration of the absolute quantifications of mRNAs, RNA half‐lives, proteins, protein half‐lives, tRNAs, with ribosome densities offers a comprehensive quantitative study of translation efficiency in the genome‐reduced bacterium
Mycoplasma pneumoniae
(
Mpn
).
Integration of the absolute quantifications of mRNAs, proteins, and protein half‐lives with ribosome densities allowed to derive the initiation and elongation rates for 475 genes (67% of all genes) in the genome‐reduced bacterium
Mpn
.
Translation initiation rate varies over 160‐fold among genes, and most of the known factors have little impact on translation efficiency.
Measured tRNA abundances vary over 100‐fold among tRNA isoacceptors, and this variation could not explain local codon elongation rates.
A comprehensive quantitative study of translation efficiency suggests the existence of unidentified mechanisms of translational regulation in
Mpn
.
The C‐terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this ...C‐terminal sequence and levels of protein expression remains unknown. Here, we identified C‐terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C‐terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels.
Synopsis
Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria.
C‐terminal amino acid biases are ubiquitous across bacterial taxonomy: positively charged residues (lysine, arginine) are enriched at the last position, while hydrophobic amino acids and threonine are depleted.
High‐throughput expression assays using a reporter gene library showed that protein expression varies up to 4‐fold, with C‐terminal positively and negatively charged residues increasing expression, and hydrophobic residues decreasing expression.
Modulation of protein degradation rate due to the identity of the C‐terminal residue could explain ˜ 85% of the variation in protein expression.
These results are relevant for the optimization of heterologous protein sequences, where the choice of C‐terminal residues could lead to increased expression levels.
Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria.
Over the past 3 years, bacterial transcriptomics has undergone a massive revolution. Increased sequencing capacity and novel tools have made it possible to explore the bacterial transcriptome to an ...unprecedented depth, which has revealed that the transcriptome is more complex and dynamic than expected. Alternative transcripts within operons challenge the classic operon definition, and many small RNAs involved in the regulation of transcription, translation and pathogenesis have been discovered. Furthermore, mRNAs may localize to specific areas in the cell, and the spatial organization and dynamics of the chromosome have been shown to be important for transcription. Epigenetic modifications of DNA also affect transcription, and RNA processing affects translation. Therefore, transcription in bacteria resembles that in eukaryotes in terms of complexity more closely than was previously thought. Here we will discuss the contribution of 'omics' approaches to these discoveries as well as the possible impact that they are expected to have in the future.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
To study basic principles of transcriptome organization in bacteria, we analyzed one of the smallest self-replicating organisms, Mycoplasma pneumoniae. We combined strand-specific tiling arrays, ...complemented by transcriptome sequencing, with more than 252 spotted arrays. We detected 117 previously undescribed, mostly noncoding transcripts, 89 of them in antisense configuration to known genes. We identified 341 operons, of which 139 are polycistronic; almost half of the latter show decaying expression in a staircase-like manner. Under various conditions, operons could be divided into 447 smaller transcriptional units, resulting in many alternative transcripts. Frequent antisense transcripts, alternative transcripts, and multiple regulators per gene imply a highly dynamic transcriptome, more similar to that of eukaryotes than previously thought.
DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt ...defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.
Proteome Organization in a Genome-Reduced Bacterium Kühner, Sebastian; van Noort, Vera; Betts, Matthew J ...
Science (American Association for the Advancement of Science),
11/2009, Letnik:
326, Številka:
5957
Journal Article
Recenzirano
The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity ...purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.
To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. ...A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and ¹³C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.
We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several
members of the KCNQ family of potassium channels. CaM ...co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in
the absence than in the presence of Ca 2+ . Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca 2+ -bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two α-helices (A and B). These
two helices encompass â¼85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of â¼130 amino acids. Within
this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1â5-10 CaM-binding
motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover,
glutathione S -transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with
KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions
to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca 2+ levels and that this interaction is disturbed when the Ca 2+ is raised. Thus, we propose that CaM acts as a mediator in the Ca 2+ -dependent modulation of KCNQ channels.
Protein post‐translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond ...genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post‐transcriptional regulatory mechanisms in the genome‐reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein‐specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N‐acetyltransferases affects protein phosphorylation, confirming cross‐talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co‐occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post‐transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.
The effect of kinase, phosphatase and N‐acetyltransferase deletions on proteome phosphorylation and acetylation was investigated in Mycoplasma pneumoniae. Bi‐directional cross‐talk between post‐transcriptional modifications suggests an underlying regulatory molecular code in prokaryotes.
Synopsis
The effect of kinase, phosphatase and N‐acetyltransferase deletions on proteome phosphorylation and acetylation was investigated in Mycoplasma pneumoniae. Bi‐directional cross‐talk between post‐transcriptional modifications suggests an underlying regulatory molecular code in prokaryotes.
Post‐translational modifications (PTMs) change the chemical properties of proteins, conferring diversity beyond the amino‐acid sequence. Proteins are often modified on multiple sites. A PTM code has been proposed, whereby modifications at specific positions influence further modifications. These regulatory circuits though have rarely been studied on a large‐scale; conservation in prokaryotes remains elusive.
Here, we studied two important PTMs– phosphorylation and lysine acetylation in the small bacterium Mycoplasma pneumoniae. We combined genetics and quantitative mass spectrometry to measure the effect of systematic kinase, phosphatase and N‐acetyltransferase deletions on proteome abundance, phosphorylation and lysine acetylation.
The data set represents a comprehensive analysis of both phosphorylation and lysine acetylation in a single prokaryote. It reveals (1) proteins often carry multiple modifications and multiple types of PTMs, reminiscent of the PTM code proposed in eukaryotes, (2) phosphorylation exerts pleiotropic effect on proteins abundances, phosphorylation, but also lysine acetylation, (3) the cross‐talk between the two PTMs is bi‐directional and (4) PTMs are frequently located at interaction interfaces and in multifunctional proteins, illustrating how PTMs could modulate protein functions affecting the way they interact.
The study provides an unbiased and quantitative view on cross‐talk between phosphorylation and lysine acetylation. It suggests that these regulatory circuits are a fundamental principle of regulation that might have evolved before the divergence of prokaryotes and eukaryotes.
Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of ...understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint‐based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time‐dependent changes, albeit using a static model. By performing an in silico knock‐out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.
A new genome‐scale metabolic reconstruction of M. pneumonia is used in combination with external metabolite measurement and protein abundance measurements to quantitatively explore the energy metabolism of this genome‐reduce human pathogen.
Synopsis
A new genome‐scale metabolic reconstruction of M. pneumonia is used in combination with external metabolite measurement and protein abundance measurements to quantitatively explore the energy metabolism of this genome‐reduce human pathogen.
We established a detailed biomass composition for M. pneumoniae, thus allowing for growth simulations.
Using our metabolic model, we corrected the metabolic network topology and the functional annotation of key metabolic enzymes.
M. pneumoniae, unlike other laboratory‐grown bacteria, uses a high fraction of energy (up to 89%) for cellular maintenance and not for growth.
Simulating different growth conditions as well as single and double mutant phenotypes, we analyzed pathway connectivity and the impact of gene deletions on the growth performance of M. pneumoniae, highlighting the limited adaptive capabilities of this minimal model organism.
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