Content
2007
Structure-function analysis of the RNA helicase maleless
17.12.2007
Nucleic Acids Research,
2008,
36 No. 3,
950-62
published on 17.12.2007
Nucleic Acids Research, online article
Nucleic Acids Research, online article
Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity.
Seven Ups the Code
14.12.2007
Patters of phosphorylation in a region of RNA polymerase II may constitute a code that controls the recruitment of regulatory factors to control gene expression. Jeffry L. Corden puts CIPSM-researcher Dirk Eick's recent work on transcription in perspective.
The development of higher forms of life would appear to have been influenced by RNA polymerase II. This enzyme transcribes the information coded by genes from DNA into messenger-RNA (mRNA), which in turn is the basis for the production of proteins. RNA polymerase II is highly conserved through evolution, with many of its structural characteristics being conserved between bacteria and humans. Single-cell organisms were already in existence 500 million years ago, with several thousand genes providing different cellular functions. Further developments seemed dependent on producing even more genes. For a highly developed organism like a human, this form of evolution would have resulted in several million genes. Researchers were therefore surprised to learn, following publication of the human genome, that a human only has around 25,000 genes – not many more than a fruit fly or a worm with approximately 15,000 to 20,000 genes. It would appear that, over the last 500 million years, other ways to produce highly complex organisms have evolved. Evolution has simply found more efficient ways to use the genes already there. But what could have made this possible? In the current issue of Science the group of Prof. Dirk Eick at the Institute of Clinical Molecular Biology and Tumor Genetics, GSF – Research Center for Environment and Health, Munich, and the group of Dr. Shona Murphy from Oxford University, England, publish results which represent a piece of the puzzle and shed new light on to the purpose of an unusual structure in RNA polymerase II. They build on earlier observations that gene expression is not just regulated by binding of the enzyme to the gene locus to which it is recruited, but also during the phase of active transcription from DNA into RNA. During this phase, parts of the newly synthesised RNA may be removed and the remaining sequences combined into new RNA message. This ‘splicing’ of RNA occurs during gene transcription, and in extreme cases, can produce RNAs coding for several thousand different proteins from a single gene.
But what was the development that permitted this advance in gene usage? The RNA polymerase II has developed a structure composed of repeats of a 7 amino-acid sequence. In humans this structure – termed “carboxyterminal domain” or CTD – is composed of 52 such repeats. It is placed exactly at the position where RNA emerges from RNA polymerase II. In less complex organisms the CTD is much shorter: a worm has 36 repeats, and yeast as few as 26, but many single-cell organisms and bacteria have never developed an obvious CTD structure.
Although the requirement of CTD for the expression of cellular genes in higher organisms is undisputed, the molecular details for the gene-specific maturation of RNAs is still largely enigmatic. The groups of Dirk Eick and Shona Murphy have now shown a differential requirement for phosphorylation of the amino acid serine at position 7 of CTD in the processing and maturation of specific gene products. These results provide the groundwork for the discovery of further pieces of the CTD puzzle and thus enlarge our knowledge of gene regulation. Given its fundamental importance, understanding the mechanism of gene regulation is essential if we are to understand cancer and other diseases at the molecular level and develop new therapies.
Publications:
-Chapman, R.D., Heidemann, M., Albert, T., Mailhammer, R., Meisterernst, M., Kremmer, E., and Eick, D. (2007) RNA polymerase II CTD is phosphorylated at serine 7 during the transcription cycle. Science, Vol. 318, Issue 5857, December 14, 2007
-Egloff, S., O’Reilly, D., Chapman, R.D., Taylor, A., Tanzhaus, K., Pitts, L., Eick, D., and Murphy, S. (2007) A specific role for serine 7 of the pol II CTD in expression of human snRNA genes. Science, Vol. 318, Issue 5857, December 14, 2007
Transcribing RNA Polymerase II Is Phosphorylated at CTD Residue Serine-7
14.12.2007
RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5′ and 3′ regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.
Serine-7 of the RNA Polymerase II CTD Is Specifically Required for snRNA Gene Expression
14.12.2007
RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene–specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type–specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.
Full View
Full View
Structure–function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement
10.12.2007
Nucleic Acids Research,
2007,
doi: 10.1093/nar/gkm1086,
676-687
published on 10.12.2007
Nucleic Acids Research, online article
Nucleic Acids Research, online article
The active center clefts of RNA polymerase (RNAP) from the archaeon Pyrococcus furiosus (Pfu) and of yeast RNAP II are nearly identical, including four protruding loops, the lid, rudder, fork 1 and fork 2. Here we present a structure–function analysis of recombinant Pfu RNAP variants lacking these cleft loops, and analyze the function of each loop at different stages of the transcription cycle. All cleft loops except fork 1 were required for promoter-directed transcription and efficient elongation. Unprimed de novo transcription required fork 2, the lid was necessary for primed initial transcription. Analysis of templates containing a pre-melted bubble showed that rewinding of upstream DNA drives RNA separation from the template. During elongation, downstream DNA strand separation required template strand binding to an invariant arginine in switch 2, and apparently interaction of an invariant arginine in fork 2 with the non-template strand.
Probing Intranuclear Environments at the Single-Molecule Level
07.12.2007
Genome activity and nuclear metabolism clearly depend on accessibility, but it is not known whether and to what extent nuclear structures limit the mobility and access of individual molecules. We used fluorescently labeled streptavidin with a nuclear localization signal as an average-sized, inert protein to probe the nuclear environment. The protein was injected into the cytoplasm of mouse cells, and single molecules were tracked in the nucleus with high-speed fluorescence microscopy. We analyzed and compared the mobility of single streptavidin molecules in structurally and functionally distinct nuclear compartments of living cells. Our results indicated that all nuclear subcompartments were easily and similarly accessible for such an average-sized protein, and even condensed heterochromatin neither excluded single molecules nor impeded their passage.The onlysignificant difference was a higher frequency of transient trappings in heterochromatin, which lasted only tens of milliseconds.The streptavidin molecules, however, did not accumulate in heterochromatin, suggesting comparatively less free volume. Interestingly, the nucleolus seemed to exclude streptavidin, as it did many other nuclear proteins,when visualized by conventional fluorescence microscopy. The tracking of single molecules, nonetheless, showed no evidence for repulsion at the border but
relatively unimpeded passage through the nucleolus. These results clearly show that single-molecule tracking can provide novel insights into mobility of proteins in the nucleus that cannot be obtained by conventional fluorescence microscopy. Our results suggest that nuclear processes may not be regulated at the level of physical accessibility but rather by local concentrationof reactants and availability of binding sites.
Full View
Full View
NC2 mobilizes TBP on core promoter TATA boxes
11.11.2007
Nature Structural & Molecular Biology,
2007,
doi:10.1038/nsmb1328,
1-6
published on 11.11.2007
www.nature.com/nsmb, online article
www.nature.com/nsmb, online article
The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box–binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Fo¨rster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP–DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA.
Full View
Full View
A mutagenesis strategy combining systematic alanine scanning with larger mutations to study protein interactions
09.11.2007
Site-directed mutagenesis (SDM) of target DNA is an invaluable tool to study protein structure–function relationships. Alanine-scanning mutagenesis has been successfully applied to systematically map functional binding epitopes. Substitution of target amino acids with alanine removes all side chain atoms past the b-carbon and does not introduce unusual backbone dihedral angle preferences. Therefore, alanine scanning is particularly useful for assessing the contribution of charged residues on the protein surface without disrupting the folding of its core. This approach requires the production of large numbers of point mutants by SDM. Numerous SDM methods have been described (reviewed in Refs.), and commercially available kits offer fast protocols based on oligonucleotide primers harboring the mutation. However, most strategies do not include a selectable marker to distinguish mutant clones from wild-type clones, and when they do so the procedure becomes significantly more laborious and time-consuming because it requires either two cycles of transformation in different bacterial strains or transfer of the target DNA sequence to and from a specialized vector. In addition, most of these procedures are not suited to generate large sequence alterations. Methods based on type IIs restriction enzymes allow precise replacement of individual nucleotides or codons and either selection for mutant clones or generation of larger mutations, but not both possibilities together. Again, when selection for mutant clones is possible, multiple restriction, ligation, fill-in, and transformation reactions are necessary and suitable restriction sites relatively close to the mutagenesis site are required. Other PCR-based approaches allow generation of point and larger mutations and rapid screening for mutant clones but involve amplification of the entire plasmid with consequent risk of introducing additional mutations. Here we describe a simple, fast, and inexpensive strategy to generate alanine substitutions as well as deletions, duplications, insertions, and larger replacements while retaining the ability to screen for mutant clones by restriction analysis.
Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells
03.11.2007
Genome integrity is constantly threatened by DNA lesions arisin from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity.
A Versatile Nanotrap for Biochemical and Functional Studies with Fluorescent Fusion Proteins
21.10.2007
Green fluorescent proteins (GFPs) and variants thereof are widely used to study protein localization and dynamics. We engineered a specific binder for fluorescent proteins based on a 13-kDa GFP binding fragment derived from a llama single chain antibody. This GFP-binding protein (GBP) can easily be produced in bacteria and coupled to a monovalent matrix. The GBP allows a fast and efficient (one-step) isolation of GFP fusion proteins and their interacting factors for biochemical analyses including mass spectroscopy and enzyme activity measurements. Moreover GBP is also suitable for chromatin immunoprecipitations from cells expressing fluorescent DNA-binding proteins. Most importantly, GBP can be fused with cellular proteins to ectopically recruit GFP fusion proteins allowing targeted manipulation of cellular structures and processes in living cells. Because of the high affinity capture of GFP fusion proteins in vitro and in vivo and a size in the lower nanometer range we refer to the immobilized GFP-binding protein as GFP-nanotrap. This versatile GFP-nanotrap enables a unique combination of microscopic, biochemical, and functional analyses with one and the same protein.
Maintenance of imprinting and nuclear architecture in cycling cells
18.09.2007
Dynamic gene repositioning has emerged as an additional level of
epigenetic gene regulation. An early example was the report of a
transient, spatial convergence (<2 micro m) of oppositely imprinted
regions (‘‘kissing’’), including the Angelman syndrome/Prader–
Willi syndrome (AS/PWS) locus and the Beckwith–Wiedemann
syndrome locus in human lymphocytes during late S phase. It was
argued that kissing is required for maintaining opposite imprints in
cycling cells. Employing 3D-FISH with a BAC contig covering the
AS/PWS region, light optical, serial sectioning, and quantitative
3D-image analysis, we observed that both loci always retained a
compact structure and did not form giant loops. Three-dimensional
distances measured among various, homologous AS/PWS segments in 393 human lymphocytes, 132 human fibroblasts, and 129 lymphoblastoid cells from Gorilla gorilla revealed a wide range of distances at any stage of interphase and in G0. At late S phase, 4% of nuclei showed distances <2 micro m, 49% showed distances >6 micro m, and 18% even showed distances >8 micro m. A similar distance variability was found for Homo sapiens (HSA) 15 centromeres in a PWS patient with a deletion of the maternal AS/PWS locus and for the Beckwith–Wiedemann syndrome loci in human lymphocytes. A transient kiss during late S phase between loci widely separated at other stages of the cell cycle seems incompatible with known global constraints of chromatin movements in cycling cells. Further experiments suggest that the previously observed convergence of AS/PWS loci during late S phase was most likely a side effect of the convergence of nucleolus organizer region-bearing acrocentric human chromosomes, including HSA 15.
Full View
Full View
