Trends in Biochemical Sciences
ReviewA roadmap for rRNA folding and assembly during transcription
Section snippets
The fate of RNA begins at birth
Many RNAs fold into stable structures that allow them to perform essential biochemical processes, such as gene regulation, splicing, and protein synthesis. A classic example is the bacterial 16S rRNA that adopts a unique structure and binds 20 or more ribosomal proteins (RPs) to form the 30S ribosomal subunit that participates in protein synthesis. Early electron micrographs of actively transcribed rDNA revealed the precursor rRNA associating with proteins as it was elongated by RNA polymerase
Folding is an unavoidable hazard during RNA synthesis
Nascent transcripts begin to fold as they emerge from the elongating RNAP, because the timescale for RNA base pairing is much shorter than the timescale for transcription (Figure 1; Box 1). Consequently, interactions between nucleotides near the 5′ end of the RNA form seconds or minutes earlier than interactions with downstream nucleotides [14,15]. It has long been thought that sequential RNA folding during transcription streamlines RNP assembly by committing the RNA to a specific subset of
Conformational switching is an evolved trait
Countless noncoding RNAs require long-range interactions spanning hundreds of nucleotides. How do such RNAs fold correctly if these long-range interactions are likely to mispair during transcription? Key insights have come from studies of riboswitches, which regulate transcription termination in response to ligand binding [24]. A number of chemical footprinting, computational, and single-molecule methods have been recently deployed to understand how riboswitches fold into structures that enable
Variable RNA folding during transcription delays protein recruitment
Folding strategies that rely only on interactions within the RNA itself become increasingly less efficient for long transcripts due to the sheer number of different structures that may form. However, misfolding of long RNAs can be circumvented by RBPs, which may capture native interactions or lower the kinetic barriers to RNA refolding [28., 29., 30.]. Moreover, assembly of a multibody RNP affords greater control over the timing of molecular interactions during RNA elongation. These
Long-range RNA interactions fall into the trap
One may ask whether certain protein binding sites are more vulnerable to variable folding than others. Interestingly, the binding sites of uS4 and uS7 both straddle long-range 16S helices 3 and 28 that close the 5′ and 3′ major domains, respectively (Figure 2A,B). This raised the possibility that stable binding is prevented by mispairing of these long-range interactions during transcription. Duss and coworkers tested this idea using single-molecule Förster resonance energy transfer (smFRET) and
Unstable complexes drive assembly
If most RPs sample the rRNA dynamically as it first starts to fold during transcription, and if too-stable local interactions can trap the rRNA in unproductive intermediates, this implies that transient non-native complexes may help kick-start ribosome assembly. How can this be? Many RPs protect their binding sites in stages [13], suggesting that the protein and RNA cofold into the native complex after they come together. For example, detailed studies of uS4 binding showed that the initial
Linking transcription elongation with RNP assembly
If proper assembly of an RNP depends on metastable structures, some transcripts may only be competent to recruit partner RBPs within a ‘window of opportunity’ that is established by the timeline of RNA synthesis (Figure 4, Key figure). Evidence for the importance of transcription speed comes from the inability to assemble functional 50S subunits when T7 RNAP was used to transcribe the 23S rRNA in E. coli cells at 37°C [45]. This defect was suppressed by growing cells at a lower temperature that
Assembly factors and modification enzymes
Ribosome assembly involves many auxiliary proteins, such as AFs, modifying enzymes, and processing enzymes (Figure 4). These factors often open or distort the rRNA structure and time the establishment of long-range interactions in the immature subunits [35]. While most bacterial AFs function after transcription and initial processing of the pre-rRNA by RNase III [52], many yeast and human AFs are core components of large processomes that assemble on the pre-rRNA during transcription [53,54].
Concluding remarks
Details of how ribosome assembly is coupled with transcription and cotranscriptional RNA folding are beginning to emerge, offering lessons for the assembly of other RBPs. Since RNA folding occurs concomitantly with synthesis and is inherently heterogenous, proteins must recognize and bind to an evolving set of RNA structures during the early stages of assembly. It is becoming clear that RNAs can adopt a range of different structures during transcription, particularly when the final structure is
Acknowledgments
We thank all the members of the Woodson laboratory for helpful discussions. M.L.R and S.A.W. are funded by the National Institutes of Health (R35 GM136351-01 to S.A.W. and K99 GM140204-01 to M.L.R.).
Declaration of interests
The authors declare no competing interests.
Glossary
- Assembly factor
- a protein that assists the assembly or maturation of ribosomal subunits but is not a component of the mature ribosome.
- Backtracking
- a type of long-lived transcriptional pause involving reverse translocation of the RNA and DNA through a secondary channel within RNAP [48]. Backtracked polymerases can be reactivated through RNA cleavage with the help of transcription factors such as GreA/GreB in bacteria and TFIIS in yeast [71].
- Helix junction
- intersection of two or more RNA double
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2022, Journal of Molecular BiologyCitation Excerpt :Bacterial ribosomal biogenesis is among the most well-studied systems exemplifying the importance of timely incorporation of cellular factors during RNP assembly. Ribosomal assembly begins during transcription, and is intimately coupled with processing and maturation of the pre-rRNA.77,78 The hierarchical addition of ribosomal proteins (RPs) to ribosomal subunits can lead to structural changes in the pre-rRNA, with each newly incorporated RP stabilizing the folded structure of its immediate RNA binding site, in turn stimulating structural changes in adjacent pre-rRNA residues that help recruit other proteins to the complex.29,30
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Twitter: @DrMaggieRodgers (M.L. Rodgers) and @woodson_lab (S.A. Woodson).