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We have studied the classic initiation elements of mRNA sequence and structure to better understand their influence on translation initiation rates in Escherichia coli. Changes introduced in the initiation codon, the Shine and Dalgarno sequence, the spacing between those two elements, and in the secondary structures within initiation domains each change the rate of 30 S ternary complex formation. We measured these differences using extension inhibition analysis, a technique we have called "toeprinting". The rate of 30 S initiation complex formation in the absence of initiation factors agrees well with in vivo translation rates in some instances, although in others a regulatory role of initiation factors in 30 S complex formation is likely. Nucleotides 5' to the Shine and Dalgarno domain facilitate ternary complex formation. 相似文献
3.
McPheeters GC 《California State Journal of Medicine》1921,19(10):411-414
4.
Maintenance of rat taste buds in primary culture 总被引:2,自引:0,他引:2
Ruiz CJ Stone LM McPheeters M Ogura T Böttger B Lasher RS Finger TE Kinnamon SC 《Chemical senses》2001,26(7):861-873
The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture. 相似文献
5.
Functional Cus1p is found with Hsh155p in a multiprotein splicing factor associated with U2 snRNA
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To explore the dynamics of snRNP structure and function, we have studied Cus1p, identified as a suppressor of U2 snRNA mutations in budding yeast. Cus1p is homologous to human SAP145, a protein present in the 17S form of the human U2 snRNP. Here, we define the Cus1p amino acids required for function in yeast. The segment of Cus1p required for binding to Hsh49p, a homolog of human SAP49, is contained within an essential region of Cus1p. Antibodies against Cus1p coimmunoprecipitate U2 snRNA, as well as Hsh155p, a protein homologous to human SAP155. Biochemical fractionation of splicing extracts and reconstitution of heat-inactivated splicing extracts from strains carrying a temperature-sensitive allele of CUS1 indicate that Cus1p and Hsh155p reside in a functional, high-salt-stable complex that is salt-dissociable from U2 snRNA. We propose that Cus1p, Hsh49p, and Hsh155p exist in a stable protein complex which can exchange with a core U2 snRNP and which is necessary for U2 snRNP function in prespliceosome assembly. The Cus1p complex shares functional as well as structural similarities with human SF3b. 相似文献
6.
Jennifer MP Woo Zhuofeng Lin Mohamad Navab Casey Van Dyck Yvette Trejo-Lopez Krystal MT Woo Hongyun Li Lawrence W Castellani Xuping Wang Noriko Iikuni Ornella J Rullo Hui Wu Antonio La Cava Alan M Fogelman Aldons J Lusis Betty P Tsao 《Arthritis research & therapy》2010,12(3):R93
Introduction
The purpose of this study was to evaluate the effects of L-4F, an apolipoprotein A-1 mimetic peptide, alone or with pravastatin, in apoE-/-Fas-/-C57BL/6 mice that spontaneously develop immunoglobulin G (IgG) autoantibodies, glomerulonephritis, osteopenia, and atherosclerotic lesions on a normal chow diet.Methods
Female mice, starting at eight to nine weeks of age, were treated for 27 weeks with 1) pravastatin, 2) L-4F, 3) L-4F plus pravastatin, or 4) vehicle control, followed by disease phenotype assessment.Results
In preliminary studies, dysfunctional, proinflammatory high-density lipoproteins (piHDL) were decreased six hours after a single L-4F, but not scrambled L-4F, injection in eight- to nine-week old mice. After 35 weeks, L-4F-treated mice, in the absence/presence of pravastatin, had significantly smaller lymph nodes and glomerular tufts (PL, LP < 0.05), lower serum levels of IgG antibodies to double stranded DNA (dsDNA) (PL < 0.05) and oxidized phospholipids (oxPLs) (PL, LP < 0.005), and elevated total and vertebral bone mineral density (PL, LP < 0.01) compared to vehicle controls. Although all treatment groups presented larger aortic root lesions compared to vehicle controls, enlarged atheromas in combination treatment mice had significantly less infiltrated CD68+ macrophages (PLP < 0.01), significantly increased mean α-actin stained area (PLP < 0.05), and significantly lower levels of circulating markers for atherosclerosis progression, CCL19 (PL, LP < 0.0005) and VCAM-1 (PL < 0.0002).Conclusions
L-4F treatment, alone or with pravastatin, significantly reduced IgG anti-dsDNA and IgG anti-oxPLs, proteinuria, glomerulonephritis, and osteopenia in a murine lupus model of accelerated atherosclerosis. Despite enlarged aortic lesions, increased smooth muscle content, decreased macrophage infiltration, and decreased pro-atherogenic chemokines in L-4F plus pravastatin treated mice suggest protective mechanisms not only on lupus-like disease, but also on potential plaque remodeling in a murine model of systemic lupus erythematosus (SLE) and accelerated atherosclerosis. 相似文献7.
Molecular architecture of the ribosome‐bound Hepatitis C Virus internal ribosomal entry site RNA
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Justus Loerke Jochen Ismer Andrea Schmidt Tarek Hilal Thiemo Sprink Kaori Yamamoto Thorsten Mielke Jörg Bürger Tanvir R Shaikh Marylena Dabrowski Peter W Hildebrand Patrick Scheerer Christian MT Spahn 《The EMBO journal》2015,34(24):3042-3058
Internal ribosomal entry sites (IRESs) are structured cis‐acting RNAs that drive an alternative, cap‐independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo‐EM reconstructions of the ribosome 80S‐ and 40S‐bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P‐site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA‐driven translation initiation. 相似文献
8.
Pseudouridine 35 (psi35) in the branch site recognition region of yeast U2 small nuclear RNA is absolutely conserved in all eukaryotes examined. Pus7p catalyzes pseudouridylation at position 35 in Saccharomyces cerevisiae U2. The pus7 deletion strain, although viable in rich medium, is growth-disadvantaged under certain conditions. To clarify the function of U2 psi35 in yeast, we used this pus7 deletion strain to screen a collection of mutant U2 small nuclear RNAs, each containing a point mutation near the branch site recognition sequence, for a synthetic growth defect phenotype. The screen identified two U2 mutants, one containing a U40 --> G40 substitution (U40G) and another having a U40 deletion (U40Delta). Yeast strains carrying either of these U2 mutations grew as well as the wild-type strain in the selection medium, but they exhibited a temperature-sensitive growth defect phenotype when coupled with the pus7 deletion (pus7Delta). A subsequent temperature shift assay and a conditional pus7 depletion (via GAL promoter shutoff) in the U2-U40 mutant genetic background caused pre-mRNA accumulation, suggesting that psi35 is required for pre-mRNA splicing under certain conditions. 相似文献
9.
Mutational analysis of the yeast U2 snRNA suggests a structural similarity to the catalytic core of group I introns. 总被引:6,自引:0,他引:6
We have used an in vitro reconstitution system to determine the effects of a large number of mutations in the highly conserved 5' terminal domain of the yeast U2 snRNA on pre-mRNA splicing. Whereas many mutations have little or no functional consequence, base substitutions in two regions were found to have drastic effects on pre-mRNA splicing. A previously unrecognized function for the U2 snRNA in the second step of splicing was found by alteration of the absolutely conserved sequence AGA upstream of the branch point recognition sequence. The effects of these mutations suggest the formation of a structure involving the U2 snRNA similar to the guanosine-binding site found in the catalytic core of group I introns. 相似文献
10.
The central nervous system first processes taste informationin the solitary nucleus, which has been almost exclusively studiedin terms of average firing rate. We analyzed interspike intervals(ISI's) of 25 taste-responsive single units in the hamster (Mesocricetusauratus) solitary nucleus. ISI's were measured during spontaneousactivity and during stimulation with NaCl, KCl, sucrose, ora mixture of the three, and graphed on semi-logarithmic plots.Two different ISI patterns were evident: simple (13 units) andcomplex (12 units). Simple ISI patterns had a single broad peakat 284.7 ± 70.4 ms spontaneous and 78.8 ± 12.8ms stimulated. All complex ISI patterns had one distinct, sharppeak for an interval about 10 ms (11.3 ± 0.4 ms: spontaneous,9.3 ± 0.5 ms: stimulated), and a second, broader peakat 273.9 ± 45.9 ms spontaneous and 71.5 ± 14.6ms stimulated. As rate of firing increased peaks in ISI patternspredictably moved towards lower intervals, but ISI pattern-typedid not change. This constancy of ISI pattern held for responsesof a unit across all stimuli and did not depend upon the stimulusspecificity or location of the unit within the rostral poleof the solitary nucleus. Apparently, the pattern that a tasteneuron generates is intrinsic to the neuron and may relate tothe way it processes tast information. 相似文献