Polysomal Ribosomes Research Articles

Ribosomal collision is not a prerequisite for ZNF598-mediated ribosome ubiquitination and disassembly of ribosomal complexes by ASCC.

Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained≥30-35 3'-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains.

Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells

Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the invitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ.

Normalized Ribo-Seq for Quantifying Absolute Global and Specific Changes in Translation.

Ribosome profiling (Ribo-Seq) is a highly sensitive method to quantify ribosome occupancies along individual mRNAs on a genome-wide scale. Hereby, ribosome-protected fragments (= footprints) are generated by nuclease digestion, isolated, and sequenced together with the corresponding randomly fragmented input samples, to determine ribosome densities (RD). For library preparation, equal amounts of total RNA are used. Subsequently, all transcript fragments are subjected to linker ligation, cDNA synthesis, and PCR amplification. Importantly, the number of reads obtained for every transcript in input and footprint samples during sequencing depends on sequencing depth and library size, as well as the relative abundance of the transcript in the sample. However, the information pertaining to the absolute amount of input and footprint sequences is lost during sample preparation, hence ruling out any conclusion whether translation is generally suppressed or activated in one condition over the other. Therefore, the RD fold-changes determined for individual genes do not reflect absolute regulation, but have to be interpreted as relative to bulk mRNA translation. Here, we modified the original ribosome profiling protocol that was first established by Ingolia et al. (2009), by adding small amounts of yeast lysate to the mammalian lysates of interest as a spike-in. This allows us to not only detect changes in the RD of specific transcripts relative to each other, but also to simultaneously measure global differences in RD (normalized ribosome density values) between samples. Graphic abstract: Global changes in translation efficiency can be detected with polysome profiling, where the proportion of polysomal ribosomes is interpreted as a proxy for ribosome density (RD) on bulk mRNA. Ribo-Seq measures changes in RD of specific mRNAs relative to bulk mRNA. The addition of a yeast-lysate, as a spike-in for normalization of read counts, allows for an absolute measurement of changes in RD.

Ribosome assembly factor Adenylate Kinase 6 maintains cell proliferation and cell size homeostasis during root growth.

Summary From the cellular perspective, organ growth is determined by production and growth of cells. Uncovering how these two processes are coordinated is essential for understanding organogenesis and regulation of organ growth.We utilized phenotypic and genetic variation of 252 natural accessions of Arabidopsis thaliana to conduct genome‐wide association studies (GWAS) for identifying genes underlying root growth variation; using a T‐DNA line candidate approach, we identified one gene involved in root growth control and characterized its function using microscopy, root growth kinematics, G2/M phase cell count, ploidy levels and ribosome polysome profiles.We identified a factor contributing to root growth control: Arabidopsis Adenylate Kinase 6 (AAK6). AAK6 is required for normal cell production and normal cell elongation, and its natural genetic variation is involved in determining root growth differences between Arabidopsis accessions. A lack of AAK6 reduces cell production in the aak6 root apex, but this is partially compensated for by longer mature root cells. Thereby, aak6 mutants exhibit compensatory cell enlargement, a phenomenon unexpected in roots. Moreover, aak6 plants accumulate 80S ribosomes while the polysome profile remains unchanged, consistent with a phenotype of perturbed ribosome biogenesis.In conclusion, AAK6 impacts ribosome abundance, cell production and thereby root growth.

US3/Rps3 controls fidelity of translation termination and programmed stop codon readthrough in co-operation with eIF3.

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

PO-108 FKBP10 is an oncofetal protein that supports lung cancer growth by promoting protein translation elongation

IntroductionCancer therapy is limited by lack of specificity. Thus, identifying molecules that are relevant for tumorigenesis and selectively expressed by cancer cells is of paramount medical importance. Here, following computational and immunohistological analyses, we found in both mouse and human that FKBP10 is an oncofetal protein that during adulthood is selectively expressed in lung cancer cells. Our results from in vitro and in vivo functional assays demonstrated that FKBP10 downregulation hinders growth of lung cancer cells and tumours owing to decreased cell proliferation. By performing proteomic analysis, we identified several members involved in protein translation as putative FKBP10 binding partners. Ribosomal profiling results indicated that FKBP10 downregulation reduces formation of ribosomal polysomes while translation initiation remains unaffected. Thus, we uncovered FKBP10 as a specific requirement of lung tumorigenesis with a role in protein translation elongation that can be exploited for therapeutic gain.Material and methods2 mouse model Immunoblotting and immunoprecipitation. shRNAs, Virus production and transduction; Measurement of 35S-labelled methionine incorporation in cells; Ribosomal profiling; CT-scan imaging.Results and discussionsIn this study, we found that FKBP10 is specifically expressed in lung tumours in mice and humans while not expressed in healthy tissues. Importantly, our data show that FKBP10 depletion hinders cancer proliferation both in vitro in human lung cancer-derived cells and in vivo in two lung cancer mouse model. The relevance of this finding is bolstered by an association analysis indicating that FKBP10 expression negatively correlates with survival of patients with lung adenocarcinoma. Here, we found that FKBP10 is present at translating ribosomes and sustains protein translation elongation and protein synthesis in lung cancer cells bearing oncogenic KRAS mutation. Importantly, FKBP10 depletion hinders proliferation of cancer cells. Thus, our results support the notion that FKBP10 is an adaptive mechanism required for the increased protein synthesis demand in proliferating lung cancer cells.ConclusionIn summary, we uncovered FKBP10 as a oncofetal protein specifically expressed in KRAS-driven lung tumours. Depletion of FKBP10 hinders tumour proliferation by a mechanisms involving suppression of protein translation elongation. Finally, we propose that inhibition of FKBP10 could be exploited for therapeutic purpouses against lung cancer and perhaps other types of malignancies.

PO-111 Capicua suppresses hepatocellular carcinoma progression by controlling ETV4-MMP1 axis

IntroductionCancer therapy is limited by lack of specificity. Thus, identifying molecules that are relevant for tumorigenesis and selectively expressed by cancer cells is of paramount medical importance. Here, following computational and immunohistological analyses, we found in both mouse and human that FKBP10 is an oncofetal protein that during adulthood is selectively expressed in lung cancer cells. Our results from in vitro and in vivo functional assays demonstrated that FKBP10 downregulation hinders growth of lung cancer cells and tumours owing to decreased cell proliferation. By performing proteomic analysis, we identified several members involved in protein translation as putative FKBP10 binding partners. Ribosomal profiling results indicated that FKBP10 downregulation reduces formation of ribosomal polysomes while translation initiation remains unaffected. Thus, we uncovered FKBP10 as a specific requirement of lung tumorigenesis with a role in protein translation elongation that can be exploited for therapeutic gain.Material and methods2 mouse model Immunoblotting and immunoprecipitation. shRNAs, Virus production and transduction; Measurement of 35S-labelled methionine incorporation in cells; Ribosomal profiling; CT-scan imaging.Results and discussionsIn this study, we found that FKBP10 is specifically expressed in lung tumours in mice and humans while not expressed in healthy tissues. Importantly, our data show that FKBP10 depletion hinders cancer proliferation both in vitro in human lung cancer-derived cells and in vivo in two lung cancer mouse model. The relevance of this finding is bolstered by an association analysis indicating that FKBP10 expression negatively correlates with survival of patients with lung adenocarcinoma. Here, we found that FKBP10 is present at translating ribosomes and sustains protein translation elongation and protein synthesis in lung cancer cells bearing oncogenic KRAS mutation. Importantly, FKBP10 depletion hinders proliferation of cancer cells. Thus, our results support the notion that FKBP10 is an adaptive mechanism required for the increased protein synthesis demand in proliferating lung cancer cells.ConclusionIn summary, we uncovered FKBP10 as a oncofetal protein specifically expressed in KRAS-driven lung tumours. Depletion of FKBP10 hinders tumour proliferation by a mechanisms involving suppression of protein translation elongation. Finally, we propose that inhibition of FKBP10 could be exploited for therapeutic purpouses against lung cancer and perhaps other types of malignancies.

Formation of new polysomes on free mRNAs in a cell-free translation systems is accompanied by partial disassembly of previously formed polysomes

A method for detection of the fluorescence-labeled mRNA in translating ribosomal complexes has been developed. It is demonstrated that in the working cell-free translation system with preformed polysomes, formation of new polysomes on free mRNA takes place. For the first time, it is shown that the process is accompanied by partial disassembly of the previously formed polysomes. This result is interpreted as an indication of the direct relationship between processes of translation termination of polysomal ribosomes and translation initiation of free mRNAs.

Conformation transitions of eukaryotic polyribosomes during multi-round translation

Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20–30 translation rounds during 6–8 h in wheat germ extract at 25°C). Three distinct types of the conformations were observed: (i) circular polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) polyribosomes and the linear (free-shaped and zigzag-like) polyribosomes were formed (‘juvenile phase’). The progressive loading of the polyribosomes with translating ribosomes induced the opening of the circular polyribosomes and the transformation of a major part of the linear polyribosomes into the dense 3D helices (‘transitional phase’). After 2 h from the beginning (about 8–10 rounds of translation) this compact form of polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes (‘steady-state phase’). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical polyribosomes.

Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3′-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5′- and 3′-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.

Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA

The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5′ and 3′ UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30–40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5′ UTR, while de novo initiation including 5′ UTR scanning proceeds at a much slower rate. Removal or replacements of 5′ and 3′ UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

Morphology of autolymphocytes used in the treatment of trophic ulcers in the setting of diabetic foot syndrome

Diabetic foot syndrome is one of the most often chronic and severe complications of diabetes mellitus. Method of local treat-ment of trophic ulcers and wounds in the setting of diabetic foot syndrome using autolymphocytes processed by leukinoferon was developed in the Institute of Clinical and Experimental Lymphology (Novossibirsk). Autolymphocytes were obtained by lymphocytapheresis on the blood cell separator «Haemonetics MCS+». The study of ultrastructural organization of identified lym-phocytes processed by leukinoferon did not reveal any disturbances of their structural integrity. Numerical density free polysomal ribosomes increased by 50%. Volumetric density of vesicular structures in cytoplasm of lymphocytes increased by 3 times. Number of excrescences and microvilli formed by plasmatic membrane increased by 2,4 times. Thus, technologies of autolymphocytes iden-tification do not result to damaged structure of cells. Processing of the lymphocytes by leukinoferon does not cause cellular damage but promotes development of structural changes.

Effect of Polidan on ultrastructural changes in the cerebral cortex and hippocampus of rats

Electron microscopy and morphometry of neurons in layer V of the somatosensory area in the neocortex and CA3 field in the dorsal hippocampus showed that single and 5-fold intraperitoneal injection of Polidan was followed by ultrastructural and metabolic changes in neurons reflecting activation of protein synthesis. The number of free ribosomes decreased; the number of polysomes and count of ribosomes in polysomes and tubules of the granular endoplasmic reticulum increased. Study of mitochondria and neuropil showed that Polidan activates synthetic processes in the neocortex and hippocampus. It should be emphasized that single treatment with Polidan led to functional activation of synthetic processes, while 5-fold injection of Polidan was followed by hyperactivation of synthetic processes and depletion of ultrastructures in the neocortex and hippocampus.

Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation

Deregulated c-myc gene expression is associated with many human and animal cancers. Myc overexpression promotes the growth of blood and lymphatic vessels, which is due in part to induction of growth factors including vascular endothelial growth factor (VEGF). We determined that the P493-6 human B-cell line increases VEGF production 10-fold upon Myc overexpression. Myc overexpression in avian B cells similarly resulted in high level VEGF production. Real-time RT-PCR analyses showed that Myc did not alter the VEGF mRNA content of these cell lines, indicating that a post-transcriptional mechanism regulates VEGF production. VEGF mRNA translation was examined by RT-PCR analysis of monosome and polysome sucrose gradient fractions from Myc-on and Myc-off P493-6 cells. Myc increased VEGF mRNA translation initiation, as VEGF mRNA loading onto polysomes increased 14-fold in Myc-on cells, and the number of ribosomes loaded per VEGF mRNA increased threefold. This translational regulation is specific to VEGF mRNA, as total polysomes show the same sucrose gradient profile in Myc-on and Myc-off cells, with no change in the percent ribosomes in polysomes, or in the number of ribosomes per polysomal mRNA. Myc stimulates VEGF production by a rapamycin- and LY294002-sensitive pathway, which does not involve alteration of eIF4E activity.

Changes in the ratio of the rough and smooth endoplasmic reticulum in fish Mauthner neurons as a measure of compensatory processes occurring in response to sensory stimulation.

Transformation of a certain proport ion of the rough endoplasmic reticulum (RER) into smooth endoplasmic reticulum (SER) is seen after a variety of experimental treatments: electrical stimulation [4J, hypoxia [1], administration of toxic substances [8], etc. The main function of the R E R is its role in protein synthesis processes, especially for proteins to be secreted from cells; free ribosomes in cytoplasmic polysomes synthesize proteins needed for maintaining viability of the cell itself, i.e., proteins for internal use [11]. Cisterns and canals of the RER lose their r ibosomes when unfavorable operat ing conditions are imposed; to a significant extent this can be explained as an adaptive response of the cell associated with the need to redirect protein synthesis to the cell's own requirements. This effect is used clinically for diagnostic purposes: increases in the numbers of free ribosomes in cells above particular levels is regarded as an indicator that pathological processes are occurring [2]. However, the events taking place in the ribosome-denuded R E R in these situations remain unstudied. The aim of the present work was to follow

Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis.

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.

Cryohomogenization of liver to make in vitro preparations of rough endoplasmic reticulum and other organelles

Cell fractionation has been used for many years to isolate organelles for biochemical study. Typically, fresh tissue is homogenized in a suitable buffer, and the various size-classes of organelles are then isolated by differential centrifugation. One of the common fractions is the microsomal fraction, which consists largely of vesiculated rough and smooth endoplasmic reticulum, and of Golgi elements.In the past we have investigated bound polysomes on the rough endoplasmic reticulum (RER) by electron microscopy, and have used cell fractionation and EM to study the orientation of ribosomes in polysomes bound to rough microsomal vesicles. However, the value of the cell fractionation material in our work has been limited by the small size of the microsomal vesicles, which are produced by vesicular fragmentation of the endoplasmic reticulum during tissue homogenization. For our work we need in vitro preparations in which the RER retains its cisternal form, rather than being fragmented into small vesicles. This report describes a method we have devised that can yield relatively intact RER in vitro.

‘Unmasking’ of stored maternal mRNAs and the activation of protein synthesis at fertilization in sea urchins

The isolation and in vitro assay of maternal mRNPs has led to differing conclusions as to whether maternal mRNAs in sea urchin eggs are in a repressed or 'masked' form. To circumvent the problems involved with in vitro approaches, we have used an in vivo assay to determine if the availability of mRNA and/or components of the translational machinery are limiting protein synthesis in the unfertilized egg. This assay involves the use of a protein synthesis elongation inhibitor to create a situation in the egg in which there is excess translational machinery available to bind mRNA. Eggs were fertilized and the rate of entry into polysomes of individual mRNAs was measured in inhibitor-treated and control embryos using 32P-labeled cDNA probes. The fraction of ribosomes in polysomes and the polysome size were also determined. The results from this in vivo approach provide strong evidence for the coactivation of both mRNAs and components of the translational machinery following fertilization. The average polysome size increases from 7.5 ribosomes per message in 15 min embryos to approximately 10.8 ribosomes in 2 h embryos. This result gives additional support to the idea that translational machinery, as well as mRNA, is activated following fertilization. We also found that individual mRNAs are recruited into polysomes with different kinetics, and that the fraction of an mRNA in polysomes in the unfertilized egg correlates with the rate at which that mRNA is recruited into polysomes following fertilization.

Protein synthesis and protein phosphorylation during heat stress, recovery, and adaptation.

Incubating cells at elevated temperatures causes an inhibition of protein synthesis. Mild heat stress at 41-42 degrees C inhibits the fraction of active, polysomal ribosomes from greater than 60% (preheating) to less than 30%. A return to 37 degrees C leads to an increase in protein synthesis, termed "recovery." Continuous incubation at 41-42 degrees C also leads to a gradual restoration of protein synthesis (greater than 70% of ribosomes reactivated by 2-4 h), termed "adaptation". Protein synthesis inhibition and reactivation is prestressed, recovered cells that contain elevated levels of the heat stress proteins occur to the same extent and at the same rate as in "naive" cells. The adaptation response requires transcription of new RNA whereas recovery does not. A large number of phosphorylation changes are induced by severe heat stress and occur with kinetics similar to the inhibition of protein synthesis. These include phosphorylation of eukaryotic protein synthesis initiation factor (eIF)-2 alpha and dephosphorylation of eIF-4B and eIF-4Fp25 (eIF-4E). However, the extent to which the modification occurs is proportional to the severity of the stress, and, under mild (41-42 degrees C) heat stress conditions, these initiation factor phosphorylation changes do not occur. Similarly, under conditions of severe heat stress eIF-2 alpha and eIF-4B frequently recover to their prestress phosphorylation state before the recovery of protein synthesis. eIF-4E dephosphorylation likewise does not occur under mild heat stress conditions. Therefore, these changes in phosphorylation states, which are thought to be sufficient cause, are not necessary for the inhibition of protein synthesis observed.

Coregulation of processing and translation: mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes.

In Escherichia coli, the final maturation of rRNA occurs in precursor particles, and recent experiments have suggested that ongoing protein synthesis may somehow be required for maturation to occur. The protein synthesis requirement for the formation of the 5' terminus of 23S rRNA has been clarified in vitro by varying the substrate of the reaction. In cell extracts, pre-23S rRNA in free ribosomes was not matured, but that in polysomes was efficiently processed. The reaction occurred in polysomes without the need for an energy source or other additives required for protein synthesis. Furthermore, when polysomes were dissociated into ribosomal subunits, they were no longer substrates for maturation; but the ribosomes became substrates again when they once more were incubated in the conditions for protein synthesis. All of these results are consistent with the notion that protein synthesis serves to form a polysomal complex that is the true substrate for maturation. Ribosomes in polysomes, possibly in the form of 70S initiation complexes, may more easily adopt a conformation that facilitates maturation cleavage. As a result, the rates of ribosome formation and protein synthesis could be coregulated.