scholarly journals Analysis of the interplay of protein biogenesis factors at the ribosome exit site reveals new role for NAC

2015 ◽  
Vol 210 (2) ◽  
pp. 287-301 ◽  
Author(s):  
Yvonne Nyathi ◽  
Martin R. Pool
Keyword(s):  
Time Window ◽  
Focal Point ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Exit Site ◽  
Α Subunit ◽  
Protein Biogenesis ◽  
Nascent Chain ◽  
Short Time Window ◽  
Short Time

The ribosome exit site is a focal point for the interaction of protein-biogenesis factors that guide the fate of nascent polypeptides. These factors include chaperones such as NAC, N-terminal-modifying enzymes like Methionine aminopeptidase (MetAP), and the signal recognition particle (SRP), which targets secretory and membrane proteins to the ER. These factors potentially compete with one another in the short time-window when the nascent chain first emerges at the exit site, suggesting a need for regulation. Here, we show that MetAP contacts the ribosome at the universal adaptor site where it is adjacent to the α subunit of NAC. SRP is also known to contact the ribosome at this site. In the absence of NAC, MetAP and SRP antagonize each other, indicating a novel role for NAC in regulating the access of MetAP and SRP to the ribosome. NAC also functions in SRP-dependent targeting and helps to protect substrates from aggregation before translocation.

scholarly journals Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

2003 ◽  
Vol 161 (4) ◽  
pp. 679-684 ◽  
Author(s):  
Ronald S. Ullers ◽  
Edith N.G. Houben ◽  
Amanda Raine ◽  
Corinne M. ten Hagen-Jongman ◽  
Måns Ehrenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Proteins ◽  
Signal Recognition Particle ◽  
Attachment Site ◽  
Trigger Factor ◽  
Signal Recognition ◽  
Exit Site ◽  
Nascent Chain ◽  
Signal Anchor

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro–synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.

scholarly journals Regulation by a chaperone improves substrate selectivity during cotranslational protein targeting

2015 ◽  
Vol 112 (25) ◽  
pp. E3169-E3178 ◽  
Author(s):  
Aileen Ariosa ◽  
Jae Ho Lee ◽  
Shuai Wang ◽  
Ishu Saraogi ◽  
Shu-ou Shan
Keyword(s):  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Critical Length ◽  
Trigger Factor ◽  
Signal Recognition ◽  
Substrate Selection ◽  
Exit Site ◽  
Protein Biogenesis ◽  
Crowded Environment ◽  
Selection Of

The ribosome exit site is a crowded environment where numerous factors contact nascent polypeptides to influence their folding, localization, and quality control. Timely and accurate selection of nascent polypeptides into the correct pathway is essential for proper protein biogenesis. To understand how this is accomplished, we probe the mechanism by which nascent polypeptides are accurately sorted between the major cotranslational chaperone trigger factor (TF) and the essential cotranslational targeting machinery, signal recognition particle (SRP). We show that TF regulates SRP function at three distinct stages, including binding of the translating ribosome, membrane targeting via recruitment of the SRP receptor, and rejection of ribosome-bound nascent polypeptides beyond a critical length. Together, these mechanisms enhance the specificity of substrate selection into both pathways. Our results reveal a multilayered mechanism of molecular interplay at the ribosome exit site, and provide a conceptual framework to understand how proteins are selected among distinct biogenesis machineries in this crowded environment.

scholarly journals 231 A SHORT TIME WINDOW TO PROFIT FROM IL-4 PLUS IL-10 ADDITION TO PROTECT CARTILAGE FROM BLOOD-INDUCED DAMAGE IN VITRO

2011 ◽  
Vol 19 ◽  
pp. S113
Author(s):  
M.E. van Meegeren ◽  
N.W. Jansen ◽  
G. Roosendaal ◽  
S.C. Mastbergen ◽  
F.P. Lafeber
Keyword(s):  
Time Window ◽  
Short Time Window ◽  
Short Time

scholarly journals N-myristoylation determines dual targeting of mammalian NADH-cytochrome b(5) reductase to ER and mitochondrial outer membranes by a mechanism of kinetic partitioning

2005 ◽  
Vol 168 (5) ◽  
pp. 735-745 ◽  
Author(s):  
Sara Colombo ◽  
Renato Longhi ◽  
Stefano Alcaro ◽  
Francesco Ortuso ◽  
Teresa Sprocati ◽  
...  
Keyword(s):  
Cytochrome B ◽  
Signal Recognition Particle ◽  
Phospholipid Bilayer ◽  
Mitochondrial Outer Membrane ◽  
Signal Recognition ◽  
Outer Membranes ◽  
Dual Targeting ◽  
Nascent Chain ◽  
Hydrophobic Amino Acids ◽  
Nadh Cytochrome

Mammalian NADH-cytochrome b(5) reductase (b5R) is an N-myristoylated protein that is dually targeted to ER and mitochondrial outer membranes. The N-linked myristate is not required for anchorage to membranes because a stretch of hydrophobic amino acids close to the NH2 terminus guarantees a tight interaction of the protein with the phospholipid bilayer. Instead, the fatty acid is required for targeting of b5R to mitochondria because a nonmyristoylated mutant is exclusively localized to the ER. Here, we have investigated the mechanism by which N-linked myristate affects b5R targeting. We find that myristoylation interferes with interaction of the nascent chain with signal recognition particle, so that a portion of the nascent chains escapes from cotranslational integration into the ER and can be post-translationally targeted to the mitochondrial outer membrane. Thus, competition between two cotranslational events, binding of signal recognition particle and modification by N-myristoylation, determines the site of translation and the localization of b5R.

scholarly journals Real-time observation of signal recognition particle binding to actively translating ribosomes

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Thomas R Noriega ◽  
Jin Chen ◽  
Peter Walter ◽  
Joseph D Puglisi
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Initial Step ◽  
Signal Recognition ◽  
Fluorescence Measurements ◽  
Nascent Chain ◽  
Time Observation

The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

scholarly journals 383. Feasibility of Specimen Self-collection in Young Children Undergoing SARS-CoV-2 Surveillance for In-person Learning

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S293-S293
Author(s):  
Jonathan Altamirano ◽  
Grace Tam ◽  
Marcela Lopez ◽  
India Robinson ◽  
Leanne Chun ◽  
...  
Keyword(s):  
Young Children ◽  
Time Window ◽  
School Administrators ◽  
Error Rates ◽  
Independent Observer ◽  
Specimen Collection ◽  
Timing Data ◽  
Short Time Window ◽  
Short Time ◽  
Over Time

Abstract Background While pediatric cases of COVID-19 are at low risk for adverse events, schoolchildren should be considered for surveillance as they can become infected at school and serve as sources of household or community transmission. Our team assessed the feasibility of young children self-collecting SARS-CoV-2 samples for surveillance testing in an educational setting. Methods Students at a K-8 school were tested weekly for SARS-CoV-2 from September 2020 - June 2021. Error rates were collected from September 2020 - January 2021. Clinical staff provided all students with instructions for anterior nares specimen self-collection and then observed them to ensure proper technique. Instructions included holding the sterile swab while making sure not to touch the tip, inserting the swab into their nostril until they start to feel resistance, and rubbing the swab in four circles before repeating the process in their other nostril. An independent observer timed random sample self-collections from April - June 2021. Results 2,590 samples were collected from 209 students during the study period when data on error rates were collected. Errors occurred in 3.3% of all student encounters (n=87). Error rates over time are shown in Figure 1, with the highest rate occurring on the first day of testing (n=20/197, 10.2%) and the lowest in January 2021 (n=1/202, 0.5%). 2,574 visits for sample self-collection occurred during the study period when independent timing data was collected (April - June 2021). Of those visits, 7.5% (n=193) were timed. The average duration of each visit was 70 seconds. Figure 1. Swab Error Rates Over Time Conclusion Pediatric self-collected lower nasal swabs are a viable and easily tolerated specimen collection method for SARS-CoV-2 surveillance in school settings, as evidenced by the low error rate and short time window of sample self-collection during testing. School administrators should expect errors to drop quickly after implementing testing. Disclosures All Authors: No reported disclosures

scholarly journals Mechanisms of membrane protein biogenesis

2014 ◽  
Vol 70 (a1) ◽  
pp. C1161-C1161
Author(s):  
Irmgard Sinning
Keyword(s):  
Membrane Proteins ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Protein Biogenesis ◽  
Cargo Proteins ◽  
Target Membrane ◽  
Hydrophobic Nature ◽  
Correct Target ◽  
Srp Rna

More than 25% of the cellular proteome comprise membrane proteins that have to be inserted into the correct target membrane. Most membrane proteins are delivered to the membrane by the signal recognition particle (SRP) pathway which relies on the recognition of an N-terminal signal sequence. In contrast to this co-translational mechanism, which avoids problems due to the hydrophobic nature of the cargo proteins, tail-anchored (TA) membrane proteins utilize a post-translational mechanism for membrane insertion – the GET pathway (guided entry of tail-anchored membrane proteins). The SRP and GET pathways are both regulated by GTP and ATP binding proteins of the SIMIBI family. However, in the SRP pathway the SRP RNA plays a unique regulatory role. Recent insights into eukaryotic SRP will be discussed.

Dual origin of the floor plate in the avian embryo

Development ◽  
2002 ◽  
Vol 129 (20) ◽  
pp. 4785-4796 ◽  
Author(s):  
Jean-Baptiste Charrier ◽  
Françoise Lapointe ◽  
Nicole M. Le Douarin ◽  
Marie-Aimée Teillet
Keyword(s):  
Time Window ◽  
Gene Expression Pattern ◽  
Floor Plate ◽  
Avian Embryo ◽  
Plate Formation ◽  
Unknown Factor ◽  
Short Time Window ◽  
Neural Ectoderm ◽  
Dual Origin ◽  
Short Time

Molecular analysis carried out on quail-chick chimeras, in which quail Hensen’s node was substituted for its chick counterpart at the five- to six-somite stage (ss), showed that the floor plate of the avian neural tube is composed of distinct areas: (1) a median one (medial floor plate or MFP) derived from Hensen’s node and characterised by the same gene expression pattern as the node cells (i.e. expression of HNF3β and Shh to the exclusion of genes early expressed in the neural ectoderm such as CSox1); and (2) lateral regions that are differentiated from the neuralised ectoderm (CSox1 positive) and form the lateral floor plate (LFP). LFP cells are induced by the MFP to express HNF3β transiently, Shh continuously and other floor-plate characteristic genes such as Netrin. In contrast to MFP cells, LFP cells also express neural markers such as Nkx2.2 and Sim1. This pattern of avian floor-plate development presents some similarities to floor-plate formation in zebrafish embryos. We also demonstrate that, although MFP and LFP have different embryonic origins in normal development, one can experimentally obtain a complete floor plate in the neural epithelium by the inductive action of either a notochord or a MFP. The competence of the neuroepithelium to respond to notochord or MFP signals is restricted to a short time window, as only the posterior-most region of the neural plate of embryos younger than 15 ss is able to differentiate a complete floor plate comprising MFP and LFP. Moreover, MFP differentiation requires between 4 and 5 days of exposure to the inducing tissues. Under the same conditions LFP and SHH-producing cells only induce LFP-type cells. These results show that the capacity to induce a complete floor plate is restricted to node-derived tissues and probably involves a still unknown factor that is not SHH, the latter being able to induce only LFP characteristics in neuralised epithelium.

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