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.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.     

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.     

Electron microscopy of polyribosomes synthesizing immunoglobin chains. Comparison with sizes determined by density-gradient-sedimentation data

Polyribosomes from the immunoglobulin-producing plasma-cell tumour X5563 were fractionated on a linear sucrose gradient and fractions containing polyribosomes carrying heavy (H) and light (L) immunoglobulin chains were examined with the electron microscope. The size distribution of the polyribosomes in the various fractions was determined. The results are consistent with previous estimates by Williamson & Askonas (1967) of the size of the polyribosomes synthesizing immunoglobulin chains, despite some discrepancies in their calculations based on sedimentation data. In particular, polyribosomes of up to 18 ribosomes were present in the part of the gradient containing H-chain polyribosomes. The polyribosomal clusters appeared to be true linear aggregates of uniformly spaced ribosomes, arranged mostly in open or zig-zag configuration. The appearance of most of them does not seem to support a general helical model of the polyribosome.     

2′-O-MODIFIED OLIGORIBONUCLEOTIDES WITH TERMINAL 3′-3′-INTERNUCLEOTIDE LINKAGE AND THEIR DERIVATIVES

A high RNA binding affinity and nuclease resistance of 2′-O-modified (2′-O-methyl, 2′-O-tetrahydropyranyl) oligoribonucleotides containing the “inverted” T at the 3′-end have been shown. The synthesis and properties of new photoactivatable perfluoroarylazide derivatives of these oligoribonucleotides are discussed.     

Differences in sequence content of nuclear and cytoplasmic polyribosomal RNA from adenovirus-infected cells.

RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.     

Binding of wheat germ ribosomes to bisulfite-modified reovirus messenger RNA: Evidence for a scanning mechanism

A scanning mechanism has been proposed (Kozak, 1978) to explain how eukaryotic ribosomes select the correct AUG codon for initiation of protein synthesis. The hypothesis is that a 40 S ribosomal subunit binds initially at or near the 5′-terminus of a message and subsequently migrates toward the interior of the messenger RNA, stopping when it encounters the first AUG codon, at which point a 60 S subunit joins and peptide bond formation begins. The scanning mechanism predicts that if a message were modified by introduction of a new AUG triplet upstream of the existing initiator codon, the adventitious AUG should be the preferred site for formation of an 80 S initiation complex. This prediction has been confirmed in the present studies with two reovirus messenger RNAs, in which sodium bisulfite was used to convert an ACG sequence (located in the 5′ untranslated region of each message) to AUG. Analysis of the ribosome-protected mRNA fragments recovered from sparsomycin-blocked 80 S initiation complexes revealed that a high percentage of wheat germ ribosomes were centered around the “unnatural” 5′-proximal AUG created by the bisulfite treatment, although some ribosomes were also positioned at the second (normal) initiator codon. The bisulfite modification was carried out in 7 m-urea at 37 °C. resulting in quantitative conversion of cytosine to uracil. Thus, both the primary and secondary structure of the message were drastically altered. These perturbations did not impair the efficiency of ribosome binding, nor did the highly unfolded state of the mRNA permit ribosomes to attach to spurious sites in the interior of the message. The data support a mechanism in which the initiator codon is selected by virtue of its position in a message (i.e. closest to the 5′-terminus), without regard to either the primary or secondary structure of the flanking regions.     

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.     

Coupling of transcription and translation in Dictyostelium discoideum nuclei

The nuclei of Dictyostelium discoideum cells have been found to contain polyribosomes active in protein synthesis. mRNA molecules enter nuclear polyribosomes while they are still being synthesized. "Non sense mediated mRNA decay" occurs in the nucleus, through the interaction of the mRNAs containing a nonsense codon with newly formed nuclear ribosomes, rather than with cytoplasmic ribosomes, as previously generally supposed.     

Analysis of ribosome binding sites from the s1 message of reovirus: Initiation at the first and second AUG codons

Two ribosome-protected initiation sites from the s1 message of reovirus have been characterized. Comparison of these sites with the previously determined sequence of s1 mRNA (Li et al., 1980) reveals that wheat germ ribosomes select and protect the first two AUG triplets in that message. This is unusual, since ribosomes initiate at a single site, the 5′-proximal AUG, in almost all other eukaryotic messenger RNAs that have been examined. The first AUG codon in s1 mRNA is preceded by a pyrimidine in position ?3, thus distinguishing it from most other eukaryotic messages, which have a purine (usually A) in that position. The behavior of s1 mRNA is consistent with the hypothesis that flanking nucleotides modulate the efficiency with which the migrating 40 S ribosomal subunit recognizes an AUG codon as a stop signal. If the first AUG triplet is flanked by suboptimal sequences, as in s1 mRNA, some 40 S ribosomes bypass that site and initiate at the next AUG downstream. The second AUG in the s1 message conforms to the consensus sequence (A-N-N-A-U-G-G) for eukaryotic initiation sites.     

Similar features in the mechanisms of mRNA translation initiation in eukaryotic and prokaryotic systems

Similar features in the mechanisms of mRNA translation initiation on prokaryotic and eukaryotic ribosomes are discussed with examples from mRNAs with nonstandard 5′-untranslated regions (5′-UTRs) and mRNAs lacking 5′-UTR (leaderless mRNAs).     

A simple general method to determine the proportion of active ribosomes in eukaryotic cells

The resistance of 80S ribosomes derived from active polyribosomes to dissociation in high KCl cone, can conveniently be used to assay the proportion of active ribosomes in eukaryotic cells. The method described has been useful in studies of organisms as diverse as fungi (yeast), protozoa (Tetrahymena), higher plants (barley), echinoderms (sea urchin), insects (Musca), birds (chick) and mammals (mouse, rat). The technique is relatively insensitive to the use of harsh mechanical treatments for cell disruption or the presence of endogenous ribonuclease activity.     

Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps

The methyl ester of m⁷G^5′ p was synthesized by a carbodiimide-catalyzed reaction of G^5′ p with methanol followed by dimethylsulfate alkylation. Comparative spectral analyses indicated that m⁷Gp · methyl ester retained the rigid conformation characteristic of the messenger RNA cap analog, m⁷G^5′ p but not its strong inhibitory activity against initiation of capped mRNA translation. Attachment of reovirus mRNA to wheat germ ribosomes, crosslinking of capbinding protein to the 5′-end of oxidized mRNA, and stimulation by this protein of capped mRNA translation in HeLa cell extract were all several-fold more sensitive to inhibition by m⁷G^5′ p than to m⁷Gp · methyl ester. Conversion of the esterified analog to m⁷G^5′ p by digestion with venom phosphodiesterase restored completely the ability to inhibit initiation complex formation. The results indicate that structural features of the 5′-terminal m⁷G cap of mRNA over and above preferred conformation are recognized during eukaryotic protein synthesis.     

Internal initiation of polyuridylic acid translation in bacterial cell-free system

The task of the present work was to answer the question: is the free 5′-end needed for effective translation of a model polyribonucleotide template — polyuridylic acid — in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5′-end and the polyuridylic acid with blocked 5′-end were compared in the bacterial cell-free translation system. To block the 5′-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5′-end and uridylic oligoribonucleotide sequence at its 3′-end, schematically described as FAM(dC)₁₀(rU)₅₀, was covalently attached (ligated) to the 5′-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5′-blocked template and on the polyuridylic acid with free 5′-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5′-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.     

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.