Intrinsic termination of transcription in Escherichia coli involves the formation of an RNA hairpin in the nascent RNA. This hairpin plays a central role in the release of the transcript and polymerase at intrinsic termination sites on the DNA template. We have created variants of the AtR2 terminator hairpin and examined the relationship between the structure and stability of this hairpin and the template positions and efficiencies of termination. The results were used to test the simple nucleic acid destabilization model of Yager and von Hippel and showed that this model must be modified to provide a distinct role for the rU-rich sequence in the nascent RNA, since a perfect palindromic sequence that is sufficiently long to form an RNA hairpin that could destabilize the entire putative 12-bp RNADNA hybrid does not trigger termination at the expected positions. Rather, our results show that both a stable terminator hairpin and the run of 6-8 rU residues that immediately follows are required for effective intrinsic termination and that termination occurs at specific and invariant template positions relative to these two components. Possible structural or kinetic modifications of the simple model are proposed in the light of these findings and of recent results implicating "inchworming" and possible conformational heterogeneity of transcription complexes in intrinsic termination. Thus, these findings argue that the structure and dimensions of the hairpin are important determinants of the termination-elongation decision and suggest that a complete mechanism is likely to involve specific interactions of the polymerase, the RNA terminator hairpin, and, perhaps, the dT-rich template sequence that codes for the run of rU residues at the 3' end of the nascent transcript.Transcription in Escherichia coli is terminated by two distinct mechanisms. One depends on rho, a hexameric protein that binds to the nascent RNA of the transcription complex and releases it at defined rho-dependent terminators along the template. The other, called intrinsic (or rho-independent) termination, also occurs at specific sites but depends only on signals encoded in the template. Intrinsic terminators all share a dyadic sequence that codes for a stable RNA hairpin followed by a string of uridine residues at the 3' end of the terminated RNA.Transcript elongation and transcript termination (followed by RNA release) represent alternative and kinetically competitive pathways for transcription at each position along the DNA template. This view of the elongation-termination decision has been formulated quantitatively in terms of competitive free energy of activation barriers (1) It has been shown for the AtR2 terminator (2) and confirmed for the AtR' terminator (Rees et al., unpublished data) that the termination pathway becomes accessible at intrinsic terminators because the transcription complex is massively destabilized in the vicinity of these sites. This provides a "zone of opportunity" for termination along the template, within which th...

Seven focus groups at a university campus were formed to identify college men's health concerns, barriers to seeking help, and recommendations to help college men adopt healthier lifestyles. Content analysis was used to identify and organize primary patterns in the focus-group data. Results of the study revealed that the college men were aware that they had important health needs but took little action to address them. The participants identified both physical and emotional health concerns. Alcohol and substance abuse were rated as the most important issues for men. The greatest barrier to seeking services was the men's socialization to be independent and conceal vulnerability. The most frequently mentioned suggestions for helping men adopt healthier lifestyles were offering health classes, providing health information call-in service, and developing a men's center. Implications of the results are discussed.

The interaction of translational elongation factor EF-G with the ribosome in the posttranslocational state has been mapped by directed hydroxyl radical probing. Localized hydroxyl radicals were generated from Fe(II) tethered to 18 different sites on the surface of EF-G bound to the ribosome. Cleavages in ribosomal RNA were mapped, providing proximity relationships between specific sites of EF-G and rRNA elements of the ribosome. Collectively, these data provide a set of constraints by which EF-G can be positioned unambiguously in the ribosome at low resolution. The proximities of different domains of EF-G to well-characterized elements of rRNA have additional implications for the mechanism of protein synthesis.

We begin by placing translocation in the overall context of the translational elongation cycle. The original two-Translation requires iterative coupled movement of mRNA site mechanism for elongation, proposed by Watson and tRNA throughout the elongation phase of protein over 30 years ago, was elaborated by the discovery of synthesis. Each new amino acid is recruited to the riboa third site, called the exit, or E site (Rheinberger et some as an aminoacyl-tRNA•EF-Tu•GTP ternary comal., 1981), and many of the details of the model were plex. Following peptide bond formation, the tRNAs and confirmed or extended. The three-site version of the associated mRNA must be translocated from one riboclassical model is summarized schematically in Figure somal site to the next in a GTP-dependent process that 1. Beginning with an initiator or peptidyl tRNA in the P is catalyzed by elongation factor EF-G. On a molecular (peptidyl) site (Figure 1A), a new aminoacyl tRNA, with scale, this movement is substantial, involving excuran anticodon that is complementary to the available A sions on the order of 50 A ˚at the elbow of tRNA during (aminoacyl)-site codon, is introduced as an aminoacyleach elongation step. Although the elongation cycle re-tRNA•EF-Tu•GTP ternary complex. Following hydrolysis quires the two G proteins (elongation factors EF-Tu and of GTP and release of EF-Tu, the aminoacyl-tRNA is EF-G) under physiological conditions, it has been shown bound to the A site (Figure 1B). The anticodon ends that protein synthesis can be carried out by the ribosome of both tRNAs interact with the 30S subunit, and their itself, in the absence of factors, or GTP, under certain acceptor (aminoacyl) ends interact with the 50S subunit. in vitro conditions (Pestka, 1969; Gavrilova et al., 1976).Attack of the peptidyl-tRNA bond by the ␣-amino group Thus, the ability to move mRNA and tRNA is an inherent of aminoacyl-tRNA, a spontaneous reaction catalyzed property of the ribosome; the factors serve to increase by peptidyl transferase (an activity of the 50S subunit), the speed and accuracy of elongation in a GTP-depenresults in peptide bond formation and transfer of the dent manner. The ribosome can therefore be considered growing peptide chain to the A-site tRNA (Figure 1C). as a macromolecular machine.Movement of the newly created peptidyl-tRNA from the Because of the fundamental importance of translation A to P site is accomplished by EF-G in a GTP-dependent to all life as we know it, and the essential similarities reaction (Figure 1D). At the same time, the deacylated between all ribosomes, the movement associated with tRNA moves to the E site. The E site is most likely located the translational elongation cycle must be one of the exclusively on the 50S subunit (Kirillov et al., 1983; Lill most ancient and basic in biology. Understanding the and Wintermeyer, 1987;Moazed and Noller, 1989a); this underlying molecular basis of this movement has prewould mean that the elaborated elongation cycle is really sented a formidable challenge to genera...

Tetracycline resistance protein Tet(O), which protects the bacterial ribosome from binding the antibiotic tetracycline, is a translational GTPase with significant similarity in both sequence and structure to the elongation factor EF-G. Here, we present an atomic model of the Tet(O)-bound 70S ribosome based on our cryo-electron microscopic reconstruction at 9.6 Å resolution. This atomic model allowed us to identify the Tet(O)-ribosome binding sites, which involve three characteristic loops in domain 4 of Tet(O). Replacements of the three-amino acid tips of these loops by a single glycine residue result in loss of Tet(O)-mediated tetracycline resistance. On the basis of these findings, the mechanism of Tet(O)-mediated tetracycline resistance can be explained in molecular detail.

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Key points• Glucocorticoids are known to be present in the developing zebrafish embryo but little is known about their physiological role at this early stage.• The zebrafish embryo demonstrates a functional glucocorticoid system from around 48 h post fertilisation.• This system and the stress response is amenable to pharmacological and genetic manipulation in a manner predicted by mammalian physiology.• Glucocorticoids play a key developmental role in hatching, swimming and stress response.• The zebrafish embryo is a relevant model for the study of glucocorticoid physiology.Abstract While glucocorticoids (GCs) are known to be present in the zebrafish embryo, little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid-related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met), an inhibitor of 11β-hydroxylase (encoded by cyp11b1), and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress-induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research.

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