| 1. |
- Quax, Tessa E. F., et al.
(författare)
-
Differential Translation Tunes Uneven Production of Operon-Encoded Proteins
- 2013
-
Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 4:5, s. 938-944
-
Tidskriftsartikel (refereegranskat)abstract
- Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.
|
|
| 2. |
- Bielen, Abraham A. M., et al.
(författare)
-
Pyrophosphate as a central energy carrier in the hydrogen-producing extremely thermophilic Caldicellulosiruptor saccharolyticus
- 2010
-
Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 307:1, s. 48-54
-
Tidskriftsartikel (refereegranskat)abstract
- The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 +/- 2 mM) and relatively low ATP levels (0.43 +/- 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticus.
|
|
| 3. |
- Blombach, Fabian, et al.
(författare)
-
Archaeal MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain
- 2014
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 462, s. 373-384
-
Tidskriftsartikel (refereegranskat)abstract
- MBF1 (multi-protein bridging factor 1) is a protein containing a conserved HTH (helix-turn-helix) domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with polyadenylated mRNA in yeast as well as in mammalian cells. aMBF1 (archaeal MBF1) is very well conserved among most archaeal lineages; however, its function has so far remained elusive. To address this, we have conducted a molecular characterization of this aMBF1. Affinity purification of interacting proteins indicates that aMBF1 binds to ribosomal subunits. On sucrose density gradients, aMBF1 co-fractionates with free 30S ribosomal subunits as well as with 70S ribosomes engaged in translation. Binding of aMBF1 to ribosomes does not inhibit translation. Using NMR spectroscopy, we show that aMBF1 contains a long intrinsically disordered linker connecting the predicted N-terminal zinc-ribbon domain with the C-terminal HTH domain. The HTH domain, which is conserved in all archaeal and eukaryotic MBF1 homologues, is directly involved in the association of aMBF1 with ribosomes. The disordered linker of the ribosome-bound aMBF1 provides the N-terminal domain with high flexibility in the aMBF1 ribosome complex. Overall, our findings suggest a role for aMBF1 in the archaeal translation process.
|
|
| 4. |
- Jore, Matthijs, et al.
(författare)
-
Structural basis for CRISPR RNA-guided DNA recognition by Cascade
- 2011
-
Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 18:5, s. 529-536
-
Tidskriftsartikel (refereegranskat)abstract
- The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.
|
|
| 5. |
- Raedts, John, et al.
(författare)
-
A Novel Bacterial Enzyme with D-Glucuronyl C5-epimerase Activity
- 2013
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:34, s. 24332-24339
-
Tidskriftsartikel (refereegranskat)abstract
- Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of D-glucuronic acid to its C5-epimer L-iduronic acid, which is essential for the function of heparan sulfate. Although L-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic D-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with D-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human D-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial D-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.
|
|
| 6. |
- van Duijn, Esther, et al.
(författare)
-
Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa
- 2012
-
Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 11:11, s. 1430-1441
-
Tidskriftsartikel (refereegranskat)abstract
- The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.
|
|
| 7. |
- Westra, Edze R, et al.
(författare)
-
H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO
- 2010
-
Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 77:6, s. 1380-1393
-
Tidskriftsartikel (refereegranskat)abstract
- The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.
|
|
| 8. |
- Charpentier, Emmanuelle, 1968-, et al.
(författare)
-
CrRNA biogenesis
- 2013
-
Ingår i: CRISPR-cas systems. - Berlin, Heidelberg : Springer Berlin/Heidelberg. - 9783642346576 - 9783642346569 - 9783642429293 ; , s. 115-144
-
Bokkapitel (refereegranskat)abstract
- Mature crRNAs are key elements in CRISPR-Cas defense against genome invaders. These short RNAs are composed of unique repeat/spacer sequences that guide the Cas protein(s) to the cognate invading nucleic acids for their destruction. The biogenesis of mature crRNAs involves highly precise processing events. Interestingly, different types of CRISPR-Cas systems have evolved distinct crRNA maturation mechanisms. The CRISPR repeat-spacer array is transcribed as a precursor CRISPR RNA molecule (pre-crRNA) that undergoes one or two maturation steps. In type I CRISPR-Cas systems, pre-crRNA is cleaved within the repeat regions by a specific Cas6-like endoribonuclease that at least in some cases is a subunit of a Cascade complex to yield the mature crRNAs. In type III systems, the standalone endoribonuclease Cas6 processes pre-crRNA by cleavage within the repeats, producing an intermediate molecule that is further trimmed to generate the mature crRNAs. Type II systems have evolved a unique crRNA biogenesis pathway, in which a trans-acting small RNA (encoded by the CRISPR-Cas locus) base pairs wiTheach repeat sequence of the pre-crRNA to form a double-stranded RNA template that is cleaved by the housekeeping endoribonuclease III in the presence of protein Cas9 (Csn1). The generated intermediates are then subjected to further maturation by a yet to be revealed mechanism. In this chapter, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in crRNA biogenesis in the three types of CRISPR-Cas systems.
|
|
| 9. |
- Makarova, Kira S, et al.
(författare)
-
Evolution and classification of the CRISPR-Cas systems
- 2011
-
Ingår i: Nature Reviews Microbiology. - : Nature Publishing Group. - 1740-1526 .- 1740-1534. ; 9:6, s. 467-477
-
Tidskriftsartikel (refereegranskat)abstract
- The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR-Cas systems and Cas proteins. Three major types of CRISPR-Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR-Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a 'polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR-cas loci.
|
|
