Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.
The UP element, a component of bacterial promoters located upstream of the ؊35 hexamer, increases transcription by interacting with the RNA polymerase ␣-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136-to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: ؊59 nnAAA(A͞T)(A͞T)T(A͞T)TTTTnnAAAAnnn ؊38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the ␣ C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the ␣-DNA interaction. Based on the evolutionary conservation of the residues in ␣ responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.Escherichia coli promoters recognized by the major form of RNA polymerase (RNAP E 70 , subunit composition ␣ 2 Ј) contain up to three recognition elements. Two elements, hexamers centered approximately 10 and 35 bp upstream of the transcription start site (1, 2), interact with 70 (3). The third element, the UP element, located upstream of the Ϫ35 hexamer, binds the C-terminal domain of the RNAP ␣-subunit (␣CTD) (4, 5). The most extensively characterized UP element is an adenine (A) and thymine (T)-rich sequence located between Ϫ40 and Ϫ60 in the rrnB P1 promoter that stimulates promoter activity at least 30-fold by increasing the initial equilibrium constant (K B ) and possibly a later step(s) in the transcription initiation pathway (k f ) (4, 6). UP elements have also been described in other promoters and can function with holoenzymes containing different factors (4, 7-11).The 8-kDa ␣CTD interacts with activator proteins as well as with DNA; the 28-kDa ␣ N-terminal domain contains determinants for dimerization, assembly with the -and Ј-subunits, and also interacts with transcription factors (4, 5, 12-15). The two domains are connected by a flexible linker, which permits the ␣CTD to bind DNA and interact with activators at different sites upstream of the core promoter (5,12,(16)(17)(18). The ␣CTD residues involved in DNA binding are highly conserved among eubacterial ␣-subunits (19, 20); therefore, the DNA sequences recognized by ␣ are also very likely to be conserved.Consensus sequences derived previously from E. coli promoters contain highly conserved Ϫ10 and Ϫ35 hexamers, but no highly conserved upstream sequences (1, 2, 21), suggesting that UP elements are not crucial for transcription of ...
We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme ␣ subunit carboxy-terminal domain (RNAP ␣CTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions −46 to −38; consensus 5-AAAAAARNR-3) stimulate transcription up to 170-fold, and the selected distal subsites (positions −57 to −47; consensus 5-AWWWWWTTTTT-3) stimulate transcription up to 16-fold. RNAP has subunit composition ␣ 2  and thus contains two copies of ␣CTD. Experiments with RNAP derivatives containing only one copy of ␣CTD indicate, in contrast to a previous report, that the two ␣CTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of ␣CTD, whereas function of the consensus distal subsite requires both copies of ␣CTD. We propose that each subsite constitutes a binding site for a copy of ␣CTD, and that binding of an ␣CTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other ␣CTD to the distal subsite.[Key Words: Promoter; RNA polymerase; ␣ subunit; UP element; transcription initiation] Received May 18, 1999; revised version accepted July 6, 1999.Bacterial promoters consist of at least three RNA polymerase (RNAP) recognition sequences: The −10 element, the −35 element, and the UP element (Hawley and McClure 1983;Ross et al. 1993). The −10 and −35 elements are recognized by the RNAP subunit (Dombroski et al. 1992), and the UP element, located upstream of the −35 element, is recognized by the RNAP ␣ subunit (Ross et al. 1993;Blatter et al. 1994). The best-characterized UP element is in the rrnB P1 promoter, in which the sequence determinants are located between positions −40 and −60 with respect to the transcription start site (Rao et al. 1994), and UP element-␣ interactions facilitate initial binding of RNAP and subsequent step(s) in transcription initiation (Rao et al. 1994;Strainic et al. 1998). A consensus UP element sequence (referred to here as the consensus full UP element), derived from binding-siteselection experiments, consists almost exclusively of A and T residues and increases promoter activity >300-fold . UP elements have been identified upstream of many bacterial and phage promoters and can function with RNAPs containing different factors (e.g., Newlands et al. 1993;Ross et al. 1993Ross et al. , 1998Fredrick et al. 1995).Each RNAP ␣ subunit consists of two domains connected by a long unstructured and/or flexible linker (Blatter et al. 1994;Jeon et al. 1997). The 28-kD aminoterminal domain (␣NTD) is responsible for dimerization of ␣ and for interaction with the remainder of RNAP (Igarashi and Ishihama 1991;Busby and Ebright 1994). The...
This investigation examined the effects of acute resistance exercise (RE), progressive resistance training (PRT), and age on the human skeletal muscle Transcriptome. Two cohorts of young and old adults [study A: 24 yr, 84 yr (n = 28); study B: 25 yr, 78 yr (n = 36)] were studied. Vastus lateralis biopsies were obtained pre- and 4 h post-RE in conjunction with the 1st and 36th (last) training session as part of a 12-wk PRT program in study A, whereas biopsies were obtained in the basal untrained state in study B. Additionally, the muscle fiber type specific (MHC I and MHC IIa) Transcriptome response to RE was examined in a subset of young and old women from study A. Transcriptome profiling was performed using HG U133 Plus 2.0 Arrays. The main findings were 1) there were 661 genes affected by RE during the 1st and 36th training bout that correlated with gains in muscle size and strength with PRT (termed the Transcriptome signature of resistance exercise adaptations); 2) the RE gene response was most pronounced in fast-twitch (MHC IIa) muscle fibers and provided additional insight into the skeletal muscle biology affected by RE; 3) skeletal muscle of young adults is more responsive to RE at the gene level compared with old adults and age also affected basal level skeletal muscle gene expression. These skeletal muscle Transcriptome findings provide further insight into the molecular basis of sarcopenia and the impact of resistance exercise at the mixed muscle and fiber type specific level.
thus presents an alternative solution to the problem of promoter selectivity.
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