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Title: Local Absence of Secondary Structure Permits Translation of mRNAs that Lack Ribosome-Binding Sites}
Article:
Abstract:
The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site( Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno–independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno–independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.
Introduction:
Shine-Dalgarno( SD) sequences reside in the 5′ untranslated region( 5′ UTR) of prokaryotic messenger RNAs and facilitate translation initiation. They act as ribosome-binding sites by recognizing a sequence motif at the 3′ end of the 16S ribosomal RNA in the 30S ribosomal subunit( referred to as anti-Shine-Dalgarno sequence, ASD) via complementary base pairing[1],[2]. The SD-ASD interaction is conserved across the prokaryotic kingdom and has even been retained in some cell organelles that evolved from prokaryotes more than a billion years ago. For example, tobacco plastids( chloroplasts) and the γ-proteobacterium Escherichia coli have identical ASD sequences in the 3′ end of their 16S ribosomal RNAs( 5′ TGGATCACCTCCTT 3′; ASD motif underlined) and, therefore, plastid SD sequences can be recognized in E. coli and vice versa[3],[4]. The SD consensus sequence is GGAGG in both systems. For efficient translation initiation to occur, the SD sequence needs to be present in the appropriate distance upstream of the start codon. The conserved spacing determined for E. coli is 4-9 nucleotides. Although the SD-dependent mechanism of translation initiation appears to be highly conserved among prokaryotes, it has long been known that an alternative mechanism of translation initiation must exist that is independent of the presence of a consensus SD sequence[5],[6]. Even some highly translated mRNAs in bacteria lack a recognizable SD motif indicating that translation initiation in the absence of an SD sequence can occur at high efficiency[6]. More recently, comparative analyses in several prokaryotes have indicated that SD-independent translation is much more widespread than previously appreciated[7]. However, how efficient translation is possible in the absence of a functional SD sequence and how faithful start codon recognition occurs is largely unknown. Here we have sought to identify sequence elements and/or structural motifs at the mRNA level that are involved in SD-independent initiation of protein biosynthesis. While we find no evidence for alternative sequence motifs or secondary structural requirements, we have discovered that mRNAs lacking an SD sequence exhibit a pronounced minimum in mRNA secondary structure at the translational start codon, suggesting that start codon accessibility is the major factor in SD-independent translation initiation. Using reporter gene constructs in E. coli, we confirm experimentally that local RNA unfoldedness is necessary and sufficient for initiation of protein biosynthesis in the absence of an SD sequence.
Discussion:
In this work, we have addressed the question how specific start codon recognition and efficient translation initiation can occur in the absence of a ribosome-binding site. Our genome-wide searches for Shine-Dalgarno-independent translation in bacterial and organellar genomes revealed that a large fraction of transcripts is translated in a Shine-Dalgarno-independent manner in all prokaryotic systems( Figure 1). The extent of Shine-Dalgarno-independent translation is variable between different groups of organisms, ranging from approximately 15% of the genes in the genome of γ-proteobacteria to 100% of the genes in mitochondrial genomes. Our data provide strong bioinformatics as well as experimental support for RNA unfoldedness being the major requirement for efficient start codon recognition in SD-independent translation initiation. At first glance, it may seem inconceivable that single-strandedness is sufficient to define an AUG triplet as initiator codon. However, it is important to realize that most of the coding sequence of mRNAs in both prokaryotes[18],[15] and eukaryotes[23] is highly structured and, therefore, not accessible in a single-stranded form. Therefore, presence of an AUG codon in an unstructed region can unambiguously define the correct translation initiation site( Figure 5). Our genome-wide analyses indicate that, at least in some prokaryotic systems, there is also a selective pressure towards start codon accessibility in SD-dependent translation( Figure 2 and Figure S1). This is consistent with the idea that the stability of mRNA folding near the SD sequence can influence translational efficiency[19] and may facilitate facile switching between SD-dependent and SD-independent translation in evolution. However, in all systems, the selective pressure towards start codon accessibility is considerably lower in mRNAs with an SD sequence than in mRNAs without an SD sequence. Moreover, it appears to be very low in cyanobacteria and entirely absent from plastids( Figure 2). Strikingly, cyanobacteria and plastids are the two systems with the by far highest prevalence of SD-independent translation( Figure 1). This suggests contrasting modes of genome-wide selection for start codon accessibility in SD-dependent translation initiation. However, for SD-independent translation, start codon accessibility appears to be a general requirement in all prokaryotic systems. It will be interesting to identify the evolutionary forces underlying these genome-wide differences in the utilization of SD-independent translation and its possible co-evolution with structural constraints in SD-dependent translation. The mechanism how the ribosome recognize the start codon in the absence of an SD sequence could be conceptually simple. The initial binding of the ribosome to the mRNA is, to a large degree, sequence independent[24]. In SD-dependent translation, the SD sequence likely mediates the subsequently occurring correct positioning of the ribosome on the mRNA. In SD-independent translation, this positioning function may be fulfilled by a single-stranded RNA region around the initiation codon. Also, single-strandedness is likely to facilitate recognition of the AUG start codon by the anticodon of the initiator tRNA-fMet. The low amount of structure in the 5′ part of the coding region of leaderless mRNAs in metazoan mitochondria([25]; Figure 2) could indicate that SD-independent translation initiation is mechanistically similar to the translation of at least some leaderless mRNAs. Translation initiation on leaderless mRNAs in animal mitochondria has been found to be independent of the presence of the large subunit of the ribosome[26]. A somewhat different mechanism may operate in kasugamycin-treated bacterial cells, where a unique type of reduced ribosomes( 61S ribosomes lacking several proteins of the small ribosomal subunit) has been shown to preferentially translate leaderless mRNAs[27]. In summary, our findings provide a plausible mechanism for start codon recognition in SD-independent translation. In addition, they should prove useful in predicting translational efficiency on a genome-wide scale and in aiding the design and optimization of transgene expression constructs in diverse groups of prokaryotes.
Protein biosynthesis ( translation ) is a highly regulated process in gene expression . In all organisms , initiation of translation depends on molecular recognition of the messenger RNA by ribosomes . In prokaryotes ( bacteria , mitochondria , and chloroplasts ) , this recognition is mediated by a specific sequence motif in the 5′ untranslated region of the mRNA , called “ribosome-binding site” or “Shine-Dalgarno sequence . ” However , many messenger RNAs lack Shine-Dalgarno sequences , and it is currently unknown how the correct translation initiation site is recognized in these mRNAs . Here , we provide insights into the mechanism of translation initiation in the absence of a ribosome-binding site . We have performed genome-wide searches for Shine-Dalgarno–independent translation in bacterial and organellar genomes and report that a large fraction of transcripts is translated in a Shine-Dalgarno–independent manner in all prokaryotic systems . We find that Shine-Dalgarno–independent translation initiation is strongly correlated with the presence of a local minimum in RNA secondary structure around the translational start codon . The significance of RNA unfoldedness as the key determinant of start codon recognition in Shine-Dalgarno–independent translation initiation was confirmed experimentally by employing reporter gene fusions in the bacterium Escherichia coli . In conclusion , our work suggests an intriguing mechanism for translation initiation on mRNAs that lack a ribosome-binding site .