Active role of elongation factor G in maintaining the mRNA reading frame during translation.

During translation, the ribosome moves along the mRNA one codon at a time with the help of elongation factor G (EF-G). Spontaneous changes in the translational reading frame are extremely rare, yet how the precise triplet-wise step is maintained is not clear. Here, we show that the ribosome is prone to spontaneous frameshifting on mRNA slippery sequences, whereas EF-G restricts frameshifting. EF-G helps to maintain the mRNA reading frame by guiding the A-site transfer RNA during translocation due to specific interactions with the tip of EF-G domain 4. Furthermore, EF-G accelerates ribosome rearrangements that restore the ribosome's control over the codon-anticodon interaction at the end of the movement. Our data explain how the mRNA reading frame is maintained during translation

Perturbation of ribosomal subunit dynamics by inhibitors of tRNA translocation

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action

HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders

Comparative Transcriptomic Analysis Identifies a Range of Immunologically Related Functional Elaborations of Lymph Node Associated Lymphatic and Blood Endothelial Cells

Lymphatic and blood vessels are formed by specialized lymphatic endothelial cells (LEC) and blood endothelial cells (BEC), respectively. These endothelial populations not only form peripheral tissue vessels, but also critical supporting structures in secondary lymphoid organs, particularly the lymph node (LN). Lymph node LEC (LN-LEC) also have been shown to have important immunological functions that are not observed in LEC from tissue lymphatics. LN-LEC can maintain peripheral tolerance through direct presentation of self-antigen via MHC-I, leading to CD8 T cell deletion; and through transfer of self-antigen to dendritic cells for presentation via MHC-II, resulting in CD4 T cell anergy. LN-LEC also can capture and archive foreign antigens, transferring them to dendritic cells for maintenance of memory CD8 T cells. The molecular basis for these functional elaborations in LN-LEC remain largely unexplored, and it is also unclear whether blood endothelial cells in LN (LN-BEC) might express similar enhanced immunologic functionality. Here, we used RNA-Seq to compare the transcriptomic profiles of freshly isolated murine LEC and BEC from LN with one another and with freshly isolated LEC from the periphery (diaphragm). We show that LN-LEC, LN-BEC, and diaphragm LEC (D-LEC) are transcriptionally distinct from one another, demonstrating both lineage and tissue-specific functional specializations. Surprisingly, tissue microenvironment differences in gene expression profiles were more numerous than those determined by endothelial cell lineage specification. In this regard, both LN-localized endothelial cell populations show a variety of functional elaborations that suggest how they may function as antigen presenting cells, and also point to as yet unexplored roles in both positive and negative regulation of innate and adaptive immune responses. The present work has defined in depth gene expression differences that point to functional specializations of endothelial cell populations in different anatomical locations, but especially the LN. Beyond the analyses provided here, these data are a resource for future work to uncover mechanisms of endothelial cell functionality

Development of corn seedlings after a period of exposition to various water potential

Fungicide treated and untreated corn seeds were sets to germinate under varying water availability conditions (0 to -12atm water potential). The seedlings obtained on each individual treatment were subsequently grown under ideal available water conditions. The results permitted to conclude that the occurrence of a water deficit during the on set of the germination process results in reductions on the length of the embryonic structures, particularly the epicotil. This effect attenuates with times due to the increase in growth velocity in environments where water potential is lower. Fungicid treated seeds, did not appear to be consistently vantageous in environments with water stress.Submetendo sementes de milho com e sem tratamento fungicida, a ambientes de germinação variáveis quanto à disponibilidade de água (0 a -12atm), o presente trabalho avaliou o desenvolvimento posterior das plântulas postas em ambientes sem limitações hídricas. Os resultados obtidos permitiram concluir que a ocorrência de déficit hídrico, durante o início do processo de germinação, promove reduções posteriores no comprimento das estruturas embrionárias e, de forma mais acentuada, do epicótilo; apesar disso, esse efeito se atenua com o passar do tempo pela elevação progressiva na velocidade de crescimento em ambientes que oferecem menores potenciais hídricos. Paralelamente, o tratamento fungicida das sementes pode não trazer vantagens em ambientes hidricamente deficientes; esta afirmação, contudo, considera o papel toalha como meio fornecedor de água o que, em contrapartida, exige cautela na sua extrapolação para as condições proporcionadas pelo solo

Structural Basis for Polyproline-Mediated Ribosome Stalling and Rescue by the Translation Elongation Factor EF-P

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyltRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation

Performance-related pay and the teaching profession: a review of the literature

This is a postprint of an article whose final and definitive form has been published in Research Papers in Education© 2002 Copyright Taylor & Francis; Research Papers in Education is available online at http://www.informaworld.comThis paper examines and summarizes research into performance-related pay. It was undertaken as part of the Teachers' Incentive Pay Project, currently in progress at the University of Exeter, which is a study of the introduction of threshold assessment and performance management for teachers in schools in England and Wales. The paper examines research into the effects of pay on employees' behaviour and considers the claimed benefits and disadvantages of performance-related pay, both generally and with particular reference to the teaching profession. Proponents of performance-related pay claim that it improves the motivation of employees and assists in the recruitment and retention of high quality staff. Disadvantages include: neglect of unrewarded tasks; disagreement about goals; competitiveness; lack of openness about failings; cost and the possibility of demotivating those who are not rewarded. Performance-related pay has long been a feature of teachers' remuneration in the US, where it has usually been promoted in response to national crises perceived to be rooted in educational failure. Traditionally, most US merit pay schemes for teachers have not been long lasting. This paper considers research into a variety of US schemes, including studies of the conditions under which they are found to succeed. Performance-related pay works best in situations in which there are easily measured outcomes, such as in manufacturing, but the outcomes of teaching are many and varied and there have been problems related to measuring teachers' effectiveness. The paper reports claims by Odden (2000) that measuring teachers' performance is now more feasible and that, therefore, the time is right for the introduction of performance-related pay for teachers

Efeito de várias disponibilidades hídricas, atuando como pré-condicionamento fisiológico durante a germinação das sementes de milho, no desenvolvimento das plântulas

The present paper considers the possible effects of physiological pre-treatment during the beginning of corn seeds germination in environments variable as to water availability (0 to -12 atm ). Periods of defficiency followed by 5 days without hydric limitations were compared with a control that remained equally, without reservations as to the disposability of water during 5 days. Results indicated that hydric pre-treatment of seeds can, if property adapted in its details, provide some advantages in the development of the seedlings derived from them. Chemical fungicide treatment of the seeds, submmited to the hydric pre-treatment may not be beneficial to the seedlings; as indicated by, in some isolated cases, the production of abnormalities in the embryonic structures.O presente trabalho buscou estimar os eventuais efeitos de pré-condicionamento fisiológico promovidos, durante o início da germinação das sementes de milho, por ambientes variáveis quanto à disponibilidade de água (0 a -12 atm). Para tanto, os períodos de deficiência eram sucedidos por prazos fixos de 5 dias sem limitações hídricas e comparados com uma testemunha que permaneceu, igualmente, sem restrições quanto à disponibilidade de água durante 5 dias. Os resultados encontrados indicaram que a técnica de pré-condicionamento hídrico das sementes pode, se devidamente adaptada em seus detalhes, propiciar algumas vantagens no desenvolvimento das plântulas delas oriundas. Adicionalmente, o tratamento químico fungicida das sementes, submetidas a pré-condicionamento hídrico, pode não trazer os benefícios esperados às plântulas; há casos isolados, ligados a produção de anormalidades nas estruturas embrionárias, em que o seu efeito é prejudicial