Structure of the chloroplast ribosome: novel domains for translation regulation

Gene expression in chloroplasts is controlled primarily through the regulation of translation. This regulation allows coordinate expression between the plastid and nuclear genomes, and is responsive to environmental conditions. Despite common ancestry with bacterial translation, chloroplast translation is more complex and involves positive regulatory mRNA elements and a host of requisite protein translation factors that do not have counterparts in bacteria. Previous proteomic analyses of the chloroplast ribosome identified a significant number of chloroplast-unique ribosomal proteins that expand upon a basic bacterial 70S-like composition. In this study, cryo-electron microscopy and single-particle reconstruction were used to calculate the structure of the chloroplast ribosome to a resolution of 15.5 Å. Chloroplast-unique proteins are visualized as novel structural additions to a basic bacterial ribosome core. These structures are located at optimal positions on the chloroplast ribosome for interaction with mRNAs during translation initiation. Visualization of these chloroplast-unique structures on the ribosome, combined with mRNA cross-linking, allows us to propose a model for translation initiation in chloroplasts in which chloroplast-unique ribosomal proteins interact with plastid-specific translation factors and RNA elements to facilitate regulated translation of chloroplast mRNAs.     

Intramolecular homologous recombination in Bacillus subtilis 168

Summary Ribosomal protein synthesis is regulated by controlling the fraction of mRNA associated with polysomes. It is known that this value changes in different developmental stages during Xenopus embryogenesis or, more generally, with changing cell growth conditions. We present here an analysis of the proportion of mRNA loaded on polysomes, carried out with probes for five different ribosomal proteins on several batches of Xenopus embryos obtained from different individuals. The results obtained indicate the existence of probe-dependent and individual differences, which reflect genetic variations in the cis- and trans-acting regulatory elements responsible for translational regulation. The fraction of ribosomal protein mRNA loaded onto polysomes can be used as an index of an individual's capacity for ribosome production.     

Structured mRNAs regulate translation initiation by binding to the platform of the ribosome

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.     

Nascent polypeptide sequences that influence ribosome function

Ribosomes catalyze protein synthesis using transfer RNAs and auxiliary proteins. Historically, ribosomes have been considered nonspecific translational machines, having no regulatory functions. However, a new class of regulatory mechanisms has been discovered that is based on interactions occurring within the ribosomal peptide exit tunnel that result in ribosome stalling during translation of an appropriate mRNA segment. These discoveries reveal an unexpectedly dynamic role ribosomes play in regulating their own activity. By using nascent leader peptides in combination with bound specific amino acids or antibiotics, ribosome functions can be altered significantly resulting in regulated expression of downstream coding regions. This review summarizes relevant findings in recent articles and outlines our current understanding of nascent peptide-induced ribosome stalling in regulating gene expression.     

A mammalian sequence-dependent upstream open reading frame mediates polyamine-regulated translation in yeast

In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.     

MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own translation before or at the formation of the first peptide bond

The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria. In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail. As in Escherichia coli , regulation takes place at the level of translation. MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E . coli , was shown to be an autoregulator of the MvaL1 operon. The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome. Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1. Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies. In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron. A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co-regulatory function of MvaL10, the homologue of the regulatory protein L10 of the β-operon of E . coli . However, we show that MvaL10 does not have a regulatory function.     

Control of rRNA expression in Escherichia coli

The control of ribosome synthesis has been a major focus in molecular biology for over 50 years. As protein synthesis is an essential, yet energetically costly, process, all cells (from bacteria to mammals) devote complex regulatory networks to fine-tune the expression of ribosomal RNA (and therefore ribosome synthesis) to the nutritional environment. In Escherichia coli, ribosomal RNA promoters are among the strongest in the cell and are regulated by trans-acting proteins (Fis and H-NS) and small molecules (guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates). Recent work has dissected many of the molecular mechanisms responsible for the strength and regulation of rRNA promoters.     

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.     

Human ribosomal protein L13a is dispensable for canonical ribosome function but indispensable for efficient rRNA methylation

Previously, we demonstrated that treatment of monocytic cells with IFN-gamma causes release of ribosomal protein L13a from the 60S ribosome and subsequent translational silencing of Ceruloplasmin (Cp) mRNA. Here, evidence using cultured cells demonstrates that Cp mRNA silencing is dependent on L13a and that L13a-deficient ribosomes are competent for global translational activity. Human monocytic U937 cells were stably transfected with two different shRNA sequences for L13a and clonally selected for more than 98% abrogation of total L13a expression. Metabolic labeling of these cells showed rescue of Cp translation from the IFN-gamma mediated translational silencing activity. Depletion of L13a caused significant reduction of methylation of ribosomal RNA and of cap-independent translation mediated by Internal Ribosome Entry Site (IRES) elements derived from p27, p53, and SNAT2 mRNAs. However, no significant differences in the ribosomal RNA processing, polysome formation, global translational activity, translational fidelity, and cell proliferation were observed between L13a-deficient and wild-type control cells. These results support the notion that ribosome can serve as a depot for releasable translation-regulatory factors unrelated to its basal polypeptide synthetic function. Unlike mammalian cells, the L13a homolog in yeast is indispensable for growth. Thus, L13a may have evolved from an essential ribosomal protein in lower eukaryotes to having a role as a dispensable extra-ribosomal function in higher eukaryotes.     

Halting a cellular production line: responses to ribosomal pausing during translation.

Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.     

Halting a cellular production line: responses to ribosomal pausing during translation

Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.     

RACK1 mRNA translation is regulated via a rapamycin-sensitive pathway and coordinated with ribosomal protein synthesis

RACK1 has been shown to interact with several proteins, this suggesting that it may play a central role in cell growth regulation. Some recent articles have described RACK1 as a component of the small ribosomal subunit. To investigate the relationship between RACK1 and ribosome, we analyzed RACK1 mRNA structure and regulation. Translational regulation was studied in HeLa cells subjected to serum or amino acid deprivation and stimulation. The results show that RACK1 mRNA has a 5' terminal oligopyrimidine sequence and that its translation is dependent on the availability of serum and amino acids in exactly the same way as any other vertebrate ribosomal protein mRNA.