We present genome-wide occupancy profiles for RNA polymerase (Pol) II, its phosphorylated forms and transcription factors in proliferating yeast. Pol II exchanges initiation factors for elongation factors during a 5′ transition that is completed 150 nucleotides downstream of the transcription start site (TSS). The resulting elongation complex is composed of all the elongation factors and shows high levels of Ser7 and Ser5 phosphorylation on the C-terminal repeat domain (CTD) of Pol II. Ser2 phosphorylation levels increase until 600-1,000 nucleotides downstream of the TSS and do not correlate with recruitment of Spt6 and Pcf11, which bind the Ser2-phosphorylated CTD in vitro. This indicates CTD-independent recruitment mechanisms and CTD masking in vivo. Elongation complexes are productive and disassemble in a two-step 3′ transition. Paf1, Spt16 (part of the FACT complex), and the CTD kinases Bur1 and Ctk1 exit upstream of the polyadenylation site, whereas Spt4, Spt5, Spt6, Spn1 (also called Iws1) and Elf1 exit downstream. Transitions are uniform and independent of gene length, type and expression.correlate with the in vivo occupancy of two factors that bind the phosphorylated CTD in vitro. General elongation complexes are active, as their gene occupancy predicts mRNA expression levels. RESULTS Genome-wide profiling reveals Pol II on a majority of genesWe determined genome-wide occupancy profiles by ChIP in exponentially growing Saccharomyces cerevisiae strains expressing tandem affinity purification (TAP)-tagged proteins (Online Methods). Chromatin immunoprecipitation was performed as described 11 , with modifications (Online Methods and Supplementary Methods). Enriched DNA fragments of an average size of 250 nucleotides (nt; Supplementary Fig. 1) were analyzed with tiling microarrays that cover the yeast genome at 4-nt resolution 12 . For data normalization, we developed a procedure that corrects for nonspecific antibody binding by using input measurements as well as mock immunoprecipitations (Supplementary Methods). Data from two or three highly reproducible replicates were averaged (Supplementary Table 1). The profile for the Pol II subunit Rpb3 (Fig. 1) matched previous profiles 13 obtained with different strains, experimental protocols and array platforms, but the new profile showed more details (Supplementary Fig. 2).Pol II was observed at genes encoding proteins, small nuclear RNA and small nucleolar RNA, and at regions producing cryptic unstable and unannotated transcripts 14 , but was lacking at genes transcribed by Pol I and Pol III (Fig. 1a and Supplementary Fig. 3). Of 4,366 yeast genes with annotated TSS and pA sites 15 , 2,465 (56%) showed Pol II peak occupancies above 20%, consistent with transcription of most of the Gene transcription begins with the assembly of Pol II and its initiation factors on promoter DNA. Pol II then starts mRNA synthesis and exchanges initiation factors for elongation factors, which are required for chromatin passage and RNA processing [1][2][3] . Whereas Pol II is unphosp...

Preparation of yeast proteinsEndogenous S. cerevisiae ten-subunit Pol II core enzyme was prepared as described 1 . An E. coli expression vector was derived from pET21b (Novagen) for the coexpression of the translational fusion of S. cerevisiae Rpb4:20 glycine linker:TFIIB and Rpb7:His 6 under the control of separate T7 promoters. Details of the vector design are available on request. Following expression in E. coli, cells were lysed by sonication in buffer A (50 mM Tris, 150 mM NaCl; pH 7.5, 0.3 mg/L leupeptin, 1.4 mg/L pepstatin A, 0.17 g/L PMSF, 0.33 g/L benzamidine and 10 mM β -mercaptoethanol). The lysate was cleared by centrifugation and applied to a Ni-NTA agarose column (Qiagen). The column was washed with buffer A containing 2 M NaCl, and the protein was eluted with a gradient of 10 mM to 200 mM imidazole in buffer A containing 150 mM NaCl. Peak fractions were diluted twofold and loaded onto a Mono-S cation exchange column (Amersham) equilibrated with buffer A containing 100 mM NaCl. The fusion protein was eluted over a total of 15 column volumes with a gradient of 0.1-1 M NaCl in buffer A. Peak fractions were concentrated and applied to a Superose 6 gel filtration column (Amersham) equilibrated with buffer B (5 mM HEPES pH 7.25, 40 mM ammonium sulfate, 10 μM ZnCl 2 , 10 mM DTT). Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. The TBP core domain (S. cerevisiae residues 61-240) expression vector was a generous gift from Dr. Sean Juo. Expression and purification of the yeast TBP core domain was as described 2 except that Superose 12 size exclusion chromatography was performed with buffer B. Peak fractions were concentrated, shock-frozen in liquid nitrogen, and stored at −80°C. 10-subunit Pol II was incubated with two molar equivalents of nucleic acid scaffold (Template, 5'-cgacacagcatcaaatgcacgatgtaacttttataggcgcccaacc;Nontemplate, 5'-ggttgggcgcctataaaagttacatcgtgcaaaatcgttatgagaa; RNA, 5'-gctgtgtcg) as described 3 and 2.5 molar equivalents of TBP. After incubation for 20 minutes at 20°C 3-5 molar equivalents of TFIIB-Rpb4/7 fusion protein were added. After incubation for 20 min. at 20°C, the complex was purified on a Superose 6 size exclusion column (Amersham). Fractions corresponding to the complex were pooled and concentrated to 4 mg/ml.Crystallization, data collection, and structure determination Crystals were grown at 20 °C using the hanging drop vapor diffusion method by mixing 1.5 µl of sample solution with 1.5 µl of reservoir solution (800 mM sodium ammonium tartrate, 100 mM HEPES pH 7.5, 5 mM DTT). Crystals were transferred stepwise to mother solution containing additionally 0-22% glycerol over 8 h, slowly cooled down to 8 °C, incubated for another 24 h, and plunged into liquid nitrogen. Diffraction data were collected in 0.75° increments at the protein crystallography beamline ID 29 at ESRF. Diffraction data were processed with XDS and scaled with XSCALE 4 . The structure was solved by molecular replacement with PHASER 5 using the first 12-subunit Pol II ...

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.