Translation of non-capped mRNAs in a eukaryotic cell-free system: acceleration of initiation rate in the course of polysome formation

Real-time monitoring of the translation of non-capped luciferase mRNA in a wheat germ cell-free system has been performed by continuous in situ measurement of the luminescence increase in the translation mixture. The phenomenon of acceleration of translation has been revealed. It has been shown that the acceleration is accompanied by the loading of translating polysomes with additional ribosomes, and thus is caused mainly by a rise in the initiation rate, rather than the stimulation of elongation or the involvement of additional mRNA molecules in translation. The acceleration requires a sufficient concentration of mRNA and depends on the sequence of the 5′ untranslated region (UTR). It can be abolished by the addition of excess cap analog (m7GpppGm). As the acceleration does not depend on the preliminary translation of other mRNAs in the same extract, the conclusion has been made that the effect is not due to activation of the ribosome population or other components of the system during translation, but rather it is the consequence of intra-polysomal events. The acceleration observed is discussed in terms of the model of two overlapping initiation pathways in eukaryotic polysomes: translation of non-capped mRNAs starts with eIF4F-independent initiation at 5′ UTR, and after the formation of sufficiently loaded polysomes, they rearrange in such a way that a mechanism of re-initiation of terminating ribosomes switches on. The eIF4F-mediated circularization of polysomes may be considered as a possible event that leads to the re-initiation switch and the resultant acceleration effect

Abstract P-27: The 30S Ribosomal Subunit Assembly Factor Rbfa Plays a Key Role in the Formation of the Central Pseudoknot and in the Correct Docking of Helix 44 of the Decoding Center

Background: Ribosome biogenesis is a complicated multi-stage process. In the cell, 30S ribosomal subunit assembly is fast and efficient, proceeding with the help of numerous assembly protein factors. The exact role of most assembly factors and mechanistic details of their operation remain unclear. The combination of genetic modification with cryo-EM analysis is widely used to identify the role of protein factors in assisting specific steps of the ribosome assembly process. The strain with knockout of a single assembly factor gene accumulates immature ribosomal particles which structural characterization reveals the information about the reactions catalyzed by the corresponding factor. Methods: We isolated the immature 30S subunits (pre-30S subunits) from the Escherichia coli strain lacking the rbfA gene (ΔrbfA) and characterized them by cryo-electron microscopy (cryo-EM). Results: Deletion of the assembly factor RbfA caused a substantial distortion of the structure of an important central pseudoknot which connects three major domains of 30S subunit and is necessary for ribosome stability. It was shown that the relative order of the assembly of the 3′ head domain and the docking of the functionally important helix 44 depends on the presence of RbfA. The formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. The cryo-EM maps for pre-30S subunits were divided into the classes corresponding to consecutive assembly intermediates: from the particles with completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and with partially distorted helix 44. Cryo-EM analysis of ΔrbfA 30S particles revealing the accumulation of two predominant classes of early and late intermediates (obtained at 2.7 Å resolutions) allowed us to suggest that RbfA participate in two stages of the 30S subunit assembly and is deeper involved in the maturation process than previously thought. Conclusion: In summary, RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including the folding of the head, and positioning of helix 44 in the decoding center at a later stage. An update to the model of factor-dependent 30S maturation was proposed, suggesting that RfbA is involved in most of the subunit assembly process

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture

Unidirectional constant rate motion of the ribosomal scanning particle during eukaryotic translation initiation

According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5′-end of mRNA and subsequently migrates along 5′-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffusion, or it is an energy-driven unidirectional movement. To address this issue, the method of continuous monitoring of protein synthesis in situ was used for high precision measurements of the times required for translation of mRNA with 5′-UTRs of different lengths and structures in mammalian and plant cell-free systems. For the first time, the relationship between the scanning time and the 5′-UTR length was determined and their linear correlation was experimentally demonstrated. The conclusion is made that the ribosome migration is an unidirectional motion with the rate being virtually independent of a particular mRNA sequence and secondary structure

Native-like Secondary Structure of Molten Globules

The most common evidence for the existence of secondary structure in a globular protein is the presence of a strongly pronounced far-UV circular dichroism (CD) spectrum. Although CD spectra of native proteins are well described and their quantitative analysis is widely used, similar studies for denatured proteins have still to be done. Far-UV CD spectra of nine proteins in the native and the pH-induced molten globule states were acquired and analyzed. Singular value decomposition showed that the spectra of molten globules could be described as a superposition of at least three independent components (most likely α-, β- and irregular structure). A self-consistent procedure of CD spectra analysis revealed the existence of a clear correlation between the shape of the molten globule spectra and the content of secondary structure elements in the corresponding native proteins, as determined from X-ray data. A mathematical expression of this correlation in terms of the Pierson coefficient amounts to the value of 0.9 for both the α-helix and the β-structure. Thus, the secondary structure of proteins in the molten globule state is close to that in the native state

Translation of non-capped mRNAs in a eukaryotic

cell-free system: acceleration of initiation rate in the course of polysome formatio

Structure and Stability of Recombinant Protein Depend on the Extra N-terminal Methionine Residue: S6 Permutein from Direct and Fusion Expression Systems

Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D 1H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence

RbfA Is Involved in Two Important Stages of 30S Subunit Assembly: Formation of the Central Pseudoknot and Docking of Helix 44 to the Decoding Center

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process