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What is the criticality of the ribosome binding site relative to the start codon in prokaryotic translation?

What is the criticality of the ribosome binding site relative to the start codon in prokaryotic translation?



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In prokaryotic translation, how critical for efficient translation is the location of the ribosome binding site, relative to the start codon?

Ideally, it is supposed to be -7b away from the start. How about if it is -9 bases away or even more? Will this have an observable effect on translation?


I found an oldish paper on this topic (from 1994). Here's a summary:

Determination of the optimal aligned spacing between the Shine-Dalgarno sequence and the translation initiation codon of Escherichia coli mRNAs. by Chen, Bjerknes, Kumar, & Jay. Nucleic Acids Research. (1994)

Experiment

The authors constructed a series of synthetic RBS regions that varied the length separating a synthetic 5-nt Shine-Dalgarno sequence from the start codon. The regions varied in size between 2 to 17 nt. They assayed the activity of a downstream enzyme, chloramphenicol acetyltransferase.

Conclusion

The authors concluded that the optimal spacing between a consensus 5-nt Shine-Dalgarno sequence (5'-GAGGT-3') and the start site was 5 nt. Note: this synthetic SD was made of the last 5 nt of a 9 nt SD consensus sequence. They also tested a synthetic SD made from the first 5 nt (5'-TAAGG-3')of the consensus SD. In this case they found the optimal distance was 9 nt.

So the optimal distance depends on where your desired SD aligns with the consensus SD sequence, which optimally is 5 nt from the start. Read on for more details.

Details

  • the RBS is considered to be large, extending 20bp on either side of a core Shine-Dalgarno (SD) sequence. These days, I often hear of the RBS spoken of in sizes that are equivalent to the SD. So in the parlance of the paper, you question is rephrased as "how does distance of the SD from the start codon effect translation?"

  • the canonical SD sequence referenced in the paper is 5'-UAAGGAGGU-3'. It is 9 nucleotides long. Distances between the SD and the start codon are defined as the number of nucleotides separate the 3' Uracil of the SD from the Adenine of the start AUG.

  • Example: the distance is 5 nt in the following mRNA

    5'… UAAGGAGGUnnnnnAUG… 3'
  • if the SD is not a complete 9 nt long, the distance is between the position of where the canonical Uracil would occur. In the following example, the distance is still calculated as 5 nt:

    5'… UAAGGAnnnnnnnnAUG… 3'
  • the average distance between the SD sequence and the start codon varies considerably and on average is 7 nt. Other investigators have found "optimal" spacing (circa 1994) ranged from 5 to 13 nt.

  • the SD site complements with region on the 16s rRNA. The start codon complements to the anticodon of fMet-tRNA loaded into the ribosomal P-site. So there are two distinct sites on the ribosome that contact the mRNA during translation initiation.

Also good to read

  • Designing Genes for Successful Protein Expression by Welch et al. Methods in Enzymology, Vol 498. (2011) (pdf)
  • Automated design of synthetic ribosome binding sites to control protein expression by Salis, Mirsky & Voigt. Nature Biotechnology. (2009). (pdf)

Note

I enjoyed researching this question because I found my own knowledge to be lacking solid empirical details. I do not have any direct experience besides this little lit review with this topic.


Ribosome

Ribosomes ( / ˈ r aɪ b ə ˌ s oʊ m , - b oʊ -/ [1] ) are macromolecular machines, found within all living cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to form polypeptide chains. Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA (rRNA) molecules and many ribosomal proteins (RPs or r-proteins). [2] [3] [4] The ribosomes and associated molecules are also known as the translational apparatus.


The Protein Synthesis Machinery

In addition to the mRNA template, many other molecules contribute to the process of translation. The composition of each component may vary across species for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNA) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors (Figure 9.4.1).

Figure 9.4.1: The protein synthesis machinery includes the large and small subunits of the ribosome, mRNA, and tRNA. (credit: modification of work by NIGMS, NIH)

In E. coli, there are 200,000 ribosomes present in every cell at any given time. A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.

Ribosomes are located in the cytoplasm in prokaryotes and in the cytoplasm and endoplasmic reticulum of eukaryotes. Ribosomes are made up of a large and a small subunit that come together for translation. The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs, a type of RNA molecule that brings amino acids to the growing chain of the polypeptide. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction.

Depending on the species, 40 to 60 types of tRNA exist in the cytoplasm. Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually &ldquotranslate&rdquo the language of RNA into the language of proteins. For each tRNA to function, it must have its specific amino acid bonded to it. In the process of tRNA &ldquocharging,&rdquo each tRNA molecule is bonded to its correct amino acid.


The Mechanism of Protein Synthesis

As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination. The process of translation is similar in prokaryotes and eukaryotes. Here we&rsquoll explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between prokaryotic and eukaryotic translation.

Initiation of Translation

Protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors (IFs IF-1, IF-2, and IF-3), and a special initiator tRNA , called ( ext_f^). The initiator tRNA interacts with the start codon AUG (or rarely, GUG), links to a formylated methionine called fMet, and can also bind IF-2. Formylated methionine is inserted by ( ext - ext_f^) at the beginning of every polypeptide chain synthesized by E. coli, but it is usually clipped off after translation is complete. When an in-frame AUG is encountered during translation elongation, a non-formylated methionine is inserted by a regular Met-tRNA Met .

In E. coli mRNA, a sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (AGGAGG), interacts with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. Guanosine triphosphate (GTP), which is a purine nucleotide triphosphate, acts as an energy source during translation&mdashboth at the start of elongation and during the ribosome&rsquos translocation.

In eukaryotes, a similar initiation complex forms, comprising mRNA, the 40S small ribosomal subunit, IFs, and nucleoside triphosphates (GTP and ATP). The charged initiator tRNA, called Met-tRNAi, does not bind fMet in eukaryotes, but is distinct from other Met-tRNAs in that it can bind IFs.

Instead of depositing at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA. A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5' cap. Once at the cap, the initiation complex tracks along the mRNA in the 5' to 3' direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. According to Kozak&rsquos rules , the nucleotides around the AUG indicate whether it is the correct start codon. Kozak&rsquos rules state that the following consensus sequence must appear around the AUG of vertebrate genes: 5'-gccRccAUGG-3'. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Essentially, the closer the sequence is to this consensus, the higher the efficiency of translation.

Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes.

Translation, Elongation, and Termination

In prokaryotes and eukaryotes, the basics of elongation are the same, so we will review elongation from the perspective of E. coli. The 50S ribosomal subunit of E. coli consists of three compartments: the A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one exception to this assembly line of tRNAs: in E. coli, ( ext - ext_f^) is capable of entering the P site directly without first entering the A site. Similarly, the eukaryotic Met-tRNAi, with help from other proteins of the initiation complex, binds directly to the P site. In both cases, this creates an initiation complex with a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

During translation elongation, the mRNA template provides specificity. As the ribosome moves along the mRNA, each mRNA codon comes into register, and specific binding with the corresponding charged tRNA anticodon is ensured. If mRNA were not present in the elongation complex, the ribosome would bind tRNAs nonspecifically.

Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon &ldquostep&rdquo of the ribosome. Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3' direction. The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase , an RNA-based enzyme that is integrated into the 50S ribosomal subunit. The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled (Figure (PageIndex<2>)). Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid protein can be translated in just 10 seconds.

Figure (PageIndex<2>): Translation begins when an initiator tRNA anticodon recognizes a codon on mRNA. The large ribosomal subunit joins the small subunit, and a second tRNA is recruited. As the mRNA moves relative to the ribosome, the polypeptide chain is formed. Entry of a release factor into the A site terminates translation and the components dissociate.

Many antibiotics inhibit bacterial protein synthesis. For example, tetracycline blocks the A site on the bacterial ribosome, and chloramphenicol blocks peptidyl transfer. What specific effect would you expect each of these antibiotics to have on protein synthesis?

Tetracycline would directly affect:

Chloramphenicol would directly affect

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released. The small and large ribosomal subunits dissociate from the mRNA and from each other they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.


Conclusions

We have studied the distribution of ribosome binding sites in 2458 completely sequenced prokaryotic genomes, in order to elucidate the possible impact of the presence and variation of RBS in translation initiation process. Our study with the publicly available NCBI data revealed that

23 % of bacterial genes lack an RBS. Also, a higher proportion of essential genes in several unipartite and multipartite genomes do not use an SD RBS. This alludes to the obligatory nature of the SD sequence and the possible adaptation of an alternate translation initiation mechanism by prokaryotes. As mRNA stability around the start codon is critical to efficient translation, the RBS spacer might be another factor to consider, other than the presence of an RBS. In addition, majority of genes with a SD sequence have motifs with a 5-10n spacer therefore, experimental analyses done to see if a change in location of such RBS motifs is detrimental may determine if this RBS spacer is optimal. Furthermore, in most cases, the distribution of SD-containing genes with respect to the COG functional categories is reflective of the relative abundance of these genes overall. However, genes with SD motifs corresponding to bins 13 and 27 appear to be mostly used in major COG group 1 (information storage and processing) and minor COG group J (translation, ribosomal structure/biogenesis) respectively. This indicates that some genes with specific COG functions may differ in their use of an SD RBS.


Obtaining Rackham RBS parts

The sequences of the Rackham RBS parts can be found in the table below. To obtain the physical DNA, we recommend two approaches -
Via de novo synthesis: Since the RBS parts are relatively short sequences, they can be easily and cheaply ordered as two single-stranded complementary oligo's and annealed. See here for a tutorial on how to construct short parts via oligo annealing.

Via the Registry distribution: The RBS parts are not yet available from the Registry distribution.


Where are ribosomes located inside a cell?

Ribosomes can function in a “free” form in the cytoplasm, called free ribosomes. However, they can also “settle” on the endoplasmic reticulum (ER) to form “rough endoplasmic reticulum (RER).” Ribosomes in the close association with the endoplasmic reticulum can facilitate the further processing of newly made proteins.

[In this figure] Ribosomes are the places where proteins were synthesized in our cells. Ribosomes can be found to be free-floating in the cytosol or associated with rough ER.


Translation

2) The 1st base of the anticodon pairs with the 3rd base of the codon and determines the number of codons recognized by the tRNA. When the anticodon is C or A, bp is specific when it is U or G, bp is less specific, and two different codons may be read by the same tRNA. When it is I, three different codons can be recognized (w/ C, U, A)

3) When an amino acid is specific by several differ codons, codons that differ in either the first two bases require different tRNAs

1) The amino acid is activated by attachment of AMP releases pyrophosphate and provides energy

2) The activated aminoacyl-adenylate remains attached to the enzyme

3) The enzyme then transfers the amino acid to the 2' or 3' OH of the ribose of the terminal adenosine on the 3' CCA tail

Hydrolysis is energetically favorable, the bond between the amino acid and the tRNA provides an energetic driving force for translation

The specific amino acid with which a tRNA is loaded is indicated with a three letter superscript like tRNA^(Met)

tRNA charging is accurate-- fewer than one error/10^4 aminoacylation events

10 members interact with different faces of tRNAs

Class I: attach amino acids to the 2' OH of the terminal ribose monomeric aminoacyl group spontaneously migrates to the 3'OH position

Class II: attaches amino acids to the 3' OH sometimes multimeric

Ten or more specific NTs may be involved in the recognition of a tRNA by its specific aminoacyl-tRNA synthetase

2) Editing site-> can accommodate the activated amino acid or the amino acid after attachment to the tRNA

Bacterial: 70S -> 50S (5S, 23S, 34 proteins) and 30S (16S, 21 proteins)

Euk: 80S -> 60S (5S, 5.8S, 28S, 49 proteins) and 40S (18S, 33 proteins)

Not really organelles because no lipid membrane more accurately large macro molecules

Each mRNA is translated simultaneously by multiple ribosomes

Can be separated on a sucrose density gradient

Deproteinized ribosome retains peptidyl transferase activity

The solvent face has great concentration of protein

Within a subunit, the proteins extends "arms" into the RNA regions. These arms are usually highly basic, and are thought to help with packing the negatively charged rRNA phosphate backbones

A site: location of aminoacyl-tRNA binding

P site: location of peptidyl-tRNA binding

E site: the exit site, occupied by the tRNA molecule released after the growing polypeptide chain is transferred to the aminoacyl-tRNA

Each tRNA starts in the A site, moves to the P site after peptide bond formation, then exits through the E site

Shine-Dalgarno sequence (aka ribosome binding sequence RBS) guides the initiating 5'-AUG to the correct position on the ribosome
- Initiation signal of 4-9 purine residues (AAGGAGG), 8-13 NTs on the 5' side of the start codon base pair with a complementary pyrimidine-rich sequence near the 3' end of the 16S rRNA of the 30S ribosomal subunit

S-D: positions the initiating 5'-AUG of the mRNA in the P site of the 30S subunit

S-D sequence can be used to initiate synthesis of more than one protein, if they are encoded in a single transcript called polycistronic mRNA

The scanning mechanism
5' cap and polyA tail bind initiation factors that form a link between the mRNA and the ribosome

Once associated with the 5' end of the mRNA, the small subunit locates the 5'-AUG start codon by scanning the RNA in the 5'-> 3' direction

- Euk tRNA and bac tRNA differ in their aminoacyl groups

- The bacterial initiator tRNA has a C-A mismatch in the acceptor stem
- The eukaryotic initiator has an A-U pair (not in internal tRNA met) in the acceptor stem that is critical for binding to EIF2-GTP

Formed in two successive reactions

In euk: all polypetides synthesized by cytosolic ribosomes begin with a Met residue (not fMet) but those synthesized by mito and chloro ribosomes begin with N-formylmethionine

1) The 30S subunit binds IF1 and IF3 preventing premature association of the 30S and 50S subunit then the 30S subunit binds the mRNA

2) The fMet-tRNA^fMet accompanied by IF-2, base-pairs with the start codon

1) Similar to IF1 and IF3 in bacteria, binding of eIF3 and eIF1A prevents premature association of ribosomal subunits (so they bind to the 40S subunit). eIF1 binds to the E site

2) Like IF2, eIF2 binds the initiator tRNA only in teh GTP bound state. eIF2 positions the initator tRNA in the P site, resulting in the formation of the 43S preinitiation complex (43S PIC)

3) eIF4E binds to 5' cap. eIF4G binds to both eIF4E and mRNA. eIF4A/4B unwind RNA helixes, forming the complex called eIF4F

4) elF4F mediates binding of the 43S preinitiaton complex to an mRNA

5) The 48S PIC scans the mRNA from 5'-> 3' in an ATP-dependent manner. ATP hydrolysis activates eIF4A/4B RNA helicase activity. During the scan, the small subunit recognizes the first start codon by base-pairing between the anticodon and AUG

6) Correct base-pairing induces conformation changes of 48S PIC, resulting in GTP hydrolysis of eIF2 and dissocation of 1,2,3,1A

7) A second GTP-regulated, initiator tRNA-binding protein eIF5B binds to the initiator tRNA, stimulating the association of 60S subunit with the small subunit


INITIATION

During translation initiation, the ribosome recruits an mRNA and selects the start codon of the open reading frame (ORF) (for recent reviews, see Milon and Rodnina 2012 Duval et al. 2015 Gualerzi and Pon 2015 see also Merrick and Pavitt 2018). In bacteria, translation initiation occurs cotranscriptionally, with the RNA polymerase (RNAP) and the ribosome physically interacting with each other (Kohler et al. 2017). The ribosome binds to the ribosome binding site (RBS) of the mRNA as soon as it emerges from the RNAP. Inhibition of translation leads to increased RNAP pausing, suggesting that transcription and translation are kinetically coupled (Landick et al. 1985 Proshkin et al. 2010). So far, almost nothing is known about the mechanism of initiation in the transcription–translation complex, a molecular machine denoted as the expressome (Kohler et al. 2017). Similarly, very little is known about initiation on mRNAs that are engaged in polysomes (Mitarai et al. 2008 Espah Borujeni and Salis 2016), as most of the mechanistic knowledge comes from studies that used free mRNAs not attached to the RNAP or to a preceding ribosome. Further studies are needed to determine whether initiation in expressomes or polysomes follows the same mechanism as initiation by the pioneering ribosome on free mRNA.

Among the different types of mRNAs found in prokaryotes, mRNAs containing the Shine–Dalgarno (SD) sequence are particularly well studied. They usually have an extended 5′ untranslated region (5′UTR) and an SD sequence located 8–10 nt upstream of the start codon (usually AUG). During SD-led initiation, the small subunit ([SSU], 30S in bacteria) is recruited to the RBS through interactions between the SD sequence and the complementary anti-SD (aSD) sequence in 16S ribosomal RNA (rRNA). Initiation on SD-led mRNAs is promoted by initiation factors IF1, IF2, and IF3. These bacterial factors display activities that resemble those of eIF1A, eIF2, and eIF1 in eukaryotes, respectively, but there is very little sequence homology between these prokaryotic and eukaryotic initiation factors. IF2 is homologous with eukaryotic initiation factor eIF5B. IF1 enhances the activities of IF2 and IF3. IF2 is a GTPase that recruits the initiator fMet-tRNA fMet . IF3 interferes with subunit association, ensures the fidelity of fMet-tRNA fMet selection over the elongator aminoacyl-tRNAs (aa-tRNAs), and helps to discriminate against mRNAs with unfavorable translation initiation regions (TIRs) (Milon and Rodnina 2012 Duval et al. 2015 Gualerzi and Pon 2015, and references therein).

However, not all mRNAs have an SD sequence. mRNAs lacking the SD sequence exist in most bacteria and archaea (Tolstrup et al. 2000 Weiner et al. 2000 Ma et al. 2002 Chang et al. 2006). The number of SD-led genes among 162 completed prokaryotic genomes varies from ∼12% to 90%, suggesting a significant number of non-SD-led or leaderless mRNAs (Chang et al. 2006). Very little is known about initiation on non-SD-led mRNAs except that the 5′UTR is usually unfolded and the AUG start codon resides in a single-stranded mRNA region (Scharff et al. 2011). In archaea and some bacteria, internal ORFs of multicistronic mRNAs are more likely to have an SD sequence than the leading ORF genes with an AUG start codon are more likely to have an SD sequence than those with GUG or UUG start codons (Ma et al. 2002 Chang et al. 2006).

Another group of mRNAs comprises leaderless mRNAs that lack a 5′UTR. Such mRNAs are widespread in a variety of bacteria (Zheng et al. 2011) and may play an important role in regulating the stress response (Grill et al. 2000 Vesper et al. 2011). A major determinant for leaderless initiation is the presence of an AUG start codon close to the 5′ end of the mRNA (Krishnan et al. 2010). Leaderless mRNAs bind to 70S ribosomes directly recruitment of fMet-tRNA fMet is facilitated by IF2 and IF3 (Grill et al. 2000 Yamamoto et al. 2016). Whereas IF2 can bind in a similar way to either the 30S subunit or 70S ribosome (Goyal et al. 2015), IF3 must move from its binding site on the 30S subunit on 50S subunit joining. Binding of IF3 to 70S ribosomes promotes their dissociation into subunits. This raises the question how IF3 can promote initiation on 70S ribosomes without splitting them into subunits. Recent results suggest that after dissociating from its 30S site on 50S subunit joining, IF3 may remain bound at the noncanonical binding site on the 50S subunit, which would allow the factor to act in leaderless initiation without promoting the dissociation of the 70S ribosome into subunits (Goyal et al. 2017). After translating the first ORF of a polycistronic mRNA, the ribosome can also reinitiate downstream at a second ORF using a 70S-scanning mechanism that requires fMet-tRNA fMet and IF3 (Yamamoto et al. 2016).

Translation initiation on SD-led mRNAs in Escherichia coli proceeds through three main assembly intermediates (Fig. 1) (Milon and Rodnina 2012 Duval et al. 2015 Gualerzi and Pon 2015). The SSU, IF1, IF2, IF3, and fMet-tRNA fMet form a labile 30S preinitiation complex (30S PIC). As soon as mRNA is recruited, start codon recognition converts the 30S PIC into the stable 30S initiation complex (30S IC). Joining of the large subunit ([LSU], 50S in bacteria) triggers the dissociation of the initiation factors, the accommodation of fMet-tRNA fMet in the P site, and the formation of the mature 70S IC, which is ready for translation elongation. The assembly pathway of the 30S PIC does not follow a strict order of factor addition. The factors can bind to the SSU independently of each other. However, there is a kinetically preferred sequence of factor association in the order IF3 and IF2, then IF1, followed by the recruitment of fMet-tRNA fMet through IF2 (Fig. 1) (Milon et al. 2012). Occasionally, fMet-tRNA fMet can form an IF2•GTP/fMet-tRNA fMet complex (Tsai et al. 2012), but this complex does not constitute an obligatory delivery pathway for fMet-tRNA fMet (Milon et al. 2010). The mRNA can bind to the SSU at any time, independent of the presence of the initiation factors (Studer and Joseph 2006 Milon et al. 2012). The association rate depends on the properties of the mRNA, such as the presence of secondary structures in the RBS, as well as the mRNA concentration (Studer and Joseph 2006). Codon recognition changes the conformation of the complex (Milon et al. 2008, 2012 Simonetti et al. 2008 Julian et al. 2011), stabilizes tRNA binding and destabilizes IF3 binding (Milon et al. 2012 Qin et al. 2012 Elvekrog and Gonzalez 2013 Hussain et al. 2016). IF3 changes its position on the ribosome in response to codon recognition (Hussain et al. 2016).

Kinetic model of translation initiation. (Top) Assembly of the 30S preinitiation complex (PIC) and 30S initiation complex (IC). Arrival times are calculated using experimentally measured bimolecular association rate constants and the in vivo concentrations of initiation factors in E. coli. Residence times are calculated from the measured dissociation rate constants of the individual components mRNA binding is shown as a last step, but can occur at any step of the assembly pathway, independent of the presence of initiation factors or fMet-tRNA fMet . Recognition of the start codon signifies the transition to the 30S IC (based on data in Milon et al. 2012). (Middle) Formation and maturation of the 70S IC. After subunit joining, IF3 may remain loosely bound to a site on the large subunit (LSU) (based on data in Goyal et al. 2017). (Bottom) Checkpoints of mRNA selection. From an mRNA-centric point of view, structured mRNAs can be recruited to the platform of the small subunit (SSU), unfold, and then accommodate in the mRNA-binding channel of the SSU (based on data in Milon et al. 2008 and Milon and Rodnina 2012).

The next major step entails the LSU docking onto the 30S IC (Fig. 1). Rapid docking depends on the presence of IF1, IF3, IF2•GTP, and fMet-tRNA fMet (Antoun et al. 2006 Milon et al. 2008 Goyal et al. 2015). In addition, the rate of subunit joining is attenuated by the mRNA depending on the sequence of the RBS, for example on the strength of the SD–aSD interactions and the length of the spacer between the SD and the start codon (Milon et al. 2008). After GTP hydrolysis by IF2, fMet-tRNA fMet accommodates in the P site (Grigoriadou et al. 2007 Milon et al. 2008 Goyal et al. 2015). Displacement of IF3 from its 30S binding site and dissociation of IF1 and IF2 from the complex allows the ribosome to make intersubunit bridges and leads to formation of the mature 70S IC (Fig. 1) (Fabbretti et al. 2007 Chen et al. 2015 Goyal et al. 2015, 2017 Liu and Fredrick 2015 MacDougall and Gonzalez 2015). The irreversible steps of start-codon recognition and GTP hydrolysis promote conformational changes of the 30S subunit and induce rotation of the two subunits relative to each other (Allen et al. 2005 Myasnikov et al. 2005 Marshall et al. 2009 Julian et al. 2011 Coureux et al. 2016 Sprink et al. 2016).

A key question is which features of the mRNA determine its translational efficiency. In bacteria, the RBS spans nucleotides –20 to +15 around the translation start codon. Translational efficiency is modulated by the nature of the codon used for initiation (AUG, GUG, or UUG), the SD sequence and the spacer between the SD sequence and the start codon, the mRNA secondary structure near the start site, and A/U-rich elements in the mRNA that are recognized by the SSU protein bS1. bS1, which is the largest and most acidic ribosomal protein, is required for the binding and unfolding of structured mRNAs (Duval et al. 2013 Byrgazov et al. 2015). The relative contribution of each specific element is not clear. The available on-line tools used to estimate translational efficiency from the thermodynamic properties of the RBS yield predictions that are quite good for engineered mRNAs (Salis et al. 2009 Kosuri et al. 2013 Reeve et al. 2014 Bonde et al. 2016). However, in natural mRNAs, each element of the RBS alone appears to have limited effect and can modulate the efficiency of initiation only within a certain context.

A more holistic approach conceptualizes the initiation pathway as comprising a sequence of kinetic checkpoints (Fig. 1) (Milon and Rodnina 2012 Duval et al. 2015 Gualerzi and Pon 2015). In this view, the initiation efficiency is determined by kinetic partitioning between the forward steps on the pathway toward the mature 70S IC, and the backward or rejection steps. The structure and thermodynamic stability of the RBS affect the association (step 1) and unfolding (step 2) of the mRNA. The identity of the start codon determines the stability of the codon–anticodon complex (step 3). Finally, the overall conformation of the 30S IC, which is modulated by the sequence context of the RBS, defines the rate of LSU joining (step 4). The kinetic model can explain any variations in the translational efficiency of different mRNAs. If the rate constants of the elemental steps are known, the translational efficiency can be predicted. In the few cases where such measurements were possible, the calculated value matched well with the directly measured translational efficiency (Milon et al. 2008, 2012). However, for most mRNAs the elemental rate constants are unknown, which hinders the use of the kinetic parameters as descriptors in global bioinformatics analysis. Although the mechanism of translation initiation is generally quite different in pro- and eukaryotes, the principles of kinetic partitioning most likely play a major role in start-site selection in eukaryotes as well (see Sokabe and Fraser 2018).


Author information

Stuart K. Archer and Nikolay E. Shirokikh: These authors contributed equally to this work.

Affiliations

Department of Genome Sciences, EMBL–Australia Collaborating Group, The John Curtin School of Medical Research, The Australian National University, Canberra, 2601, Australian Capital Territory, Australia

Stuart K. Archer, Nikolay E. Shirokikh & Thomas Preiss

Monash Bioinformatics Platform, Monash University, Melbourne, 3800, Victoria, Australia

Moscow Regional State Institute of Humanities and Social Studies, Kolomna, 140410, Russia

Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Development and Stem Cells Program, Monash University, Melbourne, 3800, Victoria, Australia

Victor Chang Cardiac Research Institute, Darlinghurst, 2010, New South Wales, Australia

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Contributions

S.K.A., T.H.B. and T.P. designed the research, S.K.A. and N.E.S. performed the experiments, S.K.A, N.E.S., T.H.B. and T.P. analysed the data, discussed the results and wrote the paper.

Corresponding author


Materials and methods

Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Escherichia coli)dnaXdoi:
10.1093/nar/14.20.8091
Uniprot ID: P06710
Gene (Human Immunodeficiency Virus Type 1)gag-poldoi:
10.1089/aid.1987.3.57
Uniprot ID: P04585
Strain, strain background (Escherichia coli)MRE600ATCCATCC #29417, (NCTC #8164, NCIB #10115)E. coli strain K-12 that lacks the RNase I activity
Strain, strain background (Escherichia coli)DH5α competent cellThermo Fisher ScientificCatalog #: 18265017
Genetic reagent (Escherichia coli)tRNAChemical BlocktRNA Phe tRNA Tyr tRNA fMet tRNA Met tRNA Glu tRNA Val tRNA Lys tRNA Arg
Genetic reagent (Escherichia coli)Total tRNA from E. coli MRE600Sigma-AldrichCatalog #: 10109541001
Transfected construct (Escherichia coli)pSP64 poly (A)PromegaCatalog #: P1241
Biological sample (Escherichia coli)ribosome (30S, 50S and 70S)doi:
10.1016/j.jmb.2007.04.042
doi:
10.1073/pnas.1520337112
Recombinant DNA reagentSacI-HFNew England BiolabsCatalog #: R3156
Recombinant DNA reagentBglIINew England BiolabsCatalog #: R0144
Recombinant DNA reagentHindIII-HFNew England BiolabsCatalog #: R3104
Recombinant DNA reagentT4 DNA ligaseNew England BiolabsCatalog #: M0202
Peptide, recombinant proteinelongation factor Tu (EF-Tu)doi:
10.1016/j.jmb.2007.04.042
Peptide, recombinant proteinelongation factor G (EF-G)doi:
10.1016/j.jmb.2007.04.042
Peptide, recombinant proteinT7 polymerasedoi:
10.1073/pnas.95.2.515
Commercial assay or kitPlasmid Miniprep SystemPromegaCatalog #: A1223
Commercial assay or kitGel and PCR Clean-Up SystemPromegaCatalog #: A9281
Commercial assay or kitDNA oligo synthesisINTEGRATED DNA TECHNOLOGIES (IDT)
Commercial assay or kitDNA sequencingACGT, INC
Chemical compound, drugpuromycinSigma-AldrichCatalog #: P8833
Chemical compound, drugcy3 maleimideClick Chemistry ToolsCatalog #: 1009
Chemical compound, drugcy5 maleimideClick Chemistry ToolsCatalog #: 1004
Chemical compound, drugPhenylalanine, L -[2,3,4,5,6-3H]-PerkinElmerCatalog #: NET112201MC
Chemical compound, drugValine, L-[U- 14 C]-PerkinElmerCatalog #: NEC291EU050UC
Chemical compound, drugMethionine, L-[ 35 S]-PerkinElmerCatalog #: NEG009T001MC
Chemical compound, drugGlutamic Acid, L-[3,4– 3 H]-PerkinElmerCatalog #: NET490001MC
Chemical compound, drugTyrosine, L
-[ring-3,5
PerkinElmerCatalog #: NET127001MC
Software, algorithmsmFRET data acquisition and analysis packageTaekjip Has laboratory website at Johns Hopkins University (http://ha.med.jhmi.edu/resources/)
Software, algorithmIDLITT, INC.
(https://www.harrisgeospatial.com/Software-Technology/IDL)
Software, algorithmHaMMyTaekjip Has laboratory website at Johns Hopkins University (http://ha.med.jhmi.edu/resources/) doi:
10.1529/biophysj.106.082487
Software, algorithmSerialEM(https://bio3d.colorado.edu/SerialEM/) doi:
10.1016/j.jsb.2005.07.007
Software, algorithmcisTEM(https://cistem.org/) doi: 10.7554/eLife.35383
Software, algorithmPhenix-1.17.1–3660(https://www.phenix-online.org/) doi:10.1107/S2059798319011471
Software, algorithmCoot v0.9 pre-ELPart of CCPEM 1.3.0 suite
(https://www.ccpem.ac.uk/index.php) doi:10.1107/S2059798317007859
Software, algorithmPyMol 2.3.2Schrödinger, LLC
(https://pymol.org)

Ribosome, EF-G, EF-Tu and tRNA preparation

tRNA fMet , tRNA Met , tRNA Phe , tRNA Val , tRNA Tyr , tRNA Lys , and tRNA Glu (purchased from Chemical Block) were aminoacylated as previously described (Lancaster and Noller, 2005 Moazed and Noller, 1989). Tight couple 70S ribosomes used for biochemical experiments and ribosomal subunit used for cryo-EM sample assembly were purified from E. coli MRE600 stain as previously described (Ermolenko et al., 2007). S6-Cy5/L9-Cy3 ribosomes were prepared by partial reconstitution of ΔS6-30S and ΔL9-50S subunits with S6-41C-Cy5 and L11-11C-Cy3 as previously described (Ermolenko et al., 2007 Ling and Ermolenko, 2015). Histidine-tagged EF-G and EF-Tu were expressed and purified using previously established procedures (Ermolenko et al., 2007).

Preparation of model mRNAs

Sequences encoding dnaX and HIV mRNAs were cloned by directional cloning downstream of T7 promoter in pSP64 plasmid vector (Promega Co). Model mRNAs (Supplementary file 1) were generated by T7 polymerase-catalyzed run-off in vitro transcription and purified by denaturing PAGE. Prior to transcription, 3’ ends of the model mRNAs were defined by linearizing the corresponding DNA templates at specific restriction sites (Supplementary file 1). smFRET measurements smFRET measurements were done as previously described (Cornish et al., 2008 Ling and Ermolenko, 2015) with modifications. The quartz slides used for total internal reflection fluorescence (TIRF) microscopy were treated with dichlorodimethylsilane (DDS) (Hua et al., 2014). The DDS surface was coated with biotinylated BSA (bio-BSA). Uncoated areas were then passivated by 0.2% Tween-20 prepared in H50 buffer which contained 20 mM HEPES (pH 7.5) and 50 mM KCl. 30 μL 0.2 mg/mL neutravidin (dissolved in H50 buffer) was bound to the biotin-BSA. For each flow-through chamber, non-specific sample binding to the slide was checked in the absence of neutravidin. Ribosomal complexes were imaged in polyamine buffer (50 mM HEPES (pH7.5), 6 mM Mg 2+ , 6 mM β-mercaptoethanol, 150 mM NH4Cl, 0.1 mM spermine and 2 mM spermidine) with 0.8 mg/mL glucose oxidase, 0.625% glucose, 1.5 mM 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic (Trolox) and 0.4 μg/mL catalase. smFRET data were acquired with 100 ms time resolution.

IDL software (ITT) was used to extract flourescence intensities of Cy3 donor (ID) and Cy5 acceptor (IA), from which apparent FRET efficiency (EFRET, hence referred as FRET) was calculated:

Traces showing single-step photobleachings for both Cy5 and Cy3 were selected using MATLAB scripts. FRET distribution histograms compiled from hundreds of smFRET traces were smoothed with a 5-point window using MATLAB and fit to two Gaussians corresponding to 0.4 and 0.6 FRET states (Ling and Ermolenko, 2015 Cornish et al., 2008 Ermolenko et al., 2007). To determine rates of fluctuations between 0.4 and 0.6 FRET states, smFRET traces were idealized by 2-state Hidden Markov model (HMM) using HaMMy software (McKinney et al., 2006).

Ribosome complexes used in smFRET experiments were assembled as follows. To fill the P site, 0.3 μM S6/L9-labeled ribosomes were incubated with 0.6 μM tRNA and 0.6 μM mRNA in polyamine buffer at 37°C for 15 min. To bind aminoacyl-tRNA to the ribosomal A site, 0.6 μM aminoacyl-tRNA were pre-incubated with 10 μM EF-Tu and 1 mM GTP in polyamine buffer at 37°C for 10 min. Then, 0.3 μM ribosomal complex containing peptidyl-tRNA in the P site was incubated with 0.6 μM aminoacyl-tRNA (complexed with EF-Tu•GTP) at 37°C for 5 min. For the mixture of all E. coli tRNAs (Figure 5—figure supplement 1), 30- (0.9 μM) or 150-fold (4.5 μM) molar excess of total aminoacyl-tRNAs (charged with all amino acids except for Tyr) were incubated with 30 nM ribosomes. After the incubation, ribosome samples were diluted to 1 nM with polyamine buffer, loaded on the slide and immobilized by neutravidin and biotinylated DNA oligo annealed to the handle sequence of the ribosome-bound model mRNA. To catalyze translocation, 1 μM EF-G•GTP was added to the imaging buffer.

To prepare dnaX_Slip mRNA-programmed ribosomes that contained N-Ac-Val-Lys-tRNA Lys in the P site (Figure 2), N-Ac-Val-tRNA Val and Lys-tRNA Lys were bound to the P and A sites of the S6/L9-labeled ribosome, respectively, as described above. After complex immobilization on the slide and removal of unbound Lys-tRNA Lys , ribosomes were incubated with 1 μM EF-G•GTP at room temperature for 10 min. Next, EF-G•GTP was replaced with the imaging buffer and a mixture of 1 μM of EF-Tu•GTP•Lys-tRNALys and 1 μM EF-G•GTP (in imaging buffer) was delivered at 0.4 mL/min speed by a syringe pump (J-Kem Scientific) after 10 s of imaging.

Puromycin assay

0.6 μM 70S ribosomes were incubated with 1.2 μM dnaX_NS mRNA and 1.2 μM N-Ac-[ 3 H]Phe-tRNA Phe in polyamine buffer at 37°C for 15 min followed by 10 min incubation with 1 mM puromycin. The puromycin reaction was terminated by diluting the ribosome samples using MgSO4-saturated 0.3 M sodium acetate (pH 5.3), and the N-Ac-[ 3 H]-Phe-puromycin was extracted ethyl acetate.

Filter-binding assay

The filter-binding assay was performed as previously described (Salsi et al., 2016 Spiegel et al., 2007) with minor modifications. Ribosome complexes were assembled with radiolabeled tRNAs ([ 14 C]Val-tRNA Val , [ 3 H]Phe-tRNA Phe , [ 3 H]Tyr-tRNA Tyr , [ 3 H]Glu-tRNA Glu and N-Ac-[ 3 H]Tyr-tRNA Tyr as indicated in figure legends) similarly to smFRET experiments described above. Ribosome complexes were applied to a nitrocellulose filter (MiliporeSigma), which was subsequently washed with 500 μl (for complexes programmed with dnaX mRNA) or 800 μl (for complexes programmed with HIV mRNA) of ice-cold polyamine buffer containing 20 mM Mg 2+ to remove unbound tRNAs. 20 mM Mg 2+ concentration was used to stabilize ribosome complexes under non-equilibrium conditions.

Frameshifting assay

0.6 μM 70S ribosomes were incubated with 1.2 μM dnaX_Slip mRNA and 1.2 μM N-Ac-Val-tRNA Val in polyamine buffer at 37°C for 15 min. The ribosomes were then incubated with 4 μM EF-G•GTP, 10 μM EF-Tu•GTP, 2.4 μM Lys-tRNA Lys , 1.2 μM Arg-tRNA Arg (binds in 0 frame) and 1.2 μM [ 3 H]Glu-tRNA Glu (binds in - one frame) at 37°C for 6 min. Incorporation of [ 3 H]Glu into the ribosome was measured by filter-binding assay as described above. Frameshifting efficiency (ribosome A-site occupancy by [ 3 H]Glu-tRNA Glu ) was normalized by the P-site occupancy of N-Ac-[ 3 H]Glu-tRNA Glu non-enzymatically bound to the ribosome programmed with dnaX_Slip ΔFSS mRNA.

HIV mRNA-70S ribosome complex assembly for cryo-EM analysis

The 70S ribosomes re-associated from 30S and 50S subunits were purified using sucrose gradient. 0.4 μM 70S ribosomes were bound with 0.7 μM N-Ac-Phe-tRNA Phe and 0.8 μM HIV_NS (GAG) mRNA in polyamine buffer.

Cryo-EM and image processing

C-flat grids (Copper, 1.2/1.3, Protochips) were glow-discharged for 30 s in a PELCO glow-discharge unit at 15 mA. 3 μl of the 70S•HIV FSS-mRNA complex at 250 nM concentration were applied to the grid and incubated for 30 s before vitrification using an FEI Vitrobot Mark IV (ThermoFisher). The grids were blotted for 3 s using blotting force 3 at 4°C and

90% humidity, plunged in liquid ethane, and stored in liquid nitrogen.

A dataset was collected using SerialEM (Mastronarde, 2005) on a Titan Krios operating at 300 kV and equipped with a K2 Summit camera (Gatan). A total of 5208 movies were collected using three shots per hole in super-resolution mode and a defocus range of −0.5 to −2.5 μm. The exposure length was 75 frames per movie yielding a total dose of 75 e-/ Å 2 . The super-resolution pixel size at the specimen level was 0.5115 Å. All movies were saved dark-corrected.

Gain and dark references were calculated using the method described by Afanasyev et al., 2015 and used to correct the collected movies in cisTEM (Grant et al., 2018). All further image processing was done using cisTEM. The movies were magnification-distortion-corrected using a calibrated distortion angle of 42.3° and a scale factor of 1.022 along the major axis and binned by a factor of 2. The movies were motion-corrected using all frames, and CTF parameters were estimated. Particles were picked using the particle picker tool in cisTEM and then curated manually. A total of 640,261 particles were extracted in 648 2 pixel boxes.

Extracted particles were aligned to an unpublished reference volume using a global search in the resolution range from 8 to 300 Å (for classification workflow, see Figure 8—figure supplement 1). The resulting 3D reconstruction was calculated using 50% of the particles with the highest scores and had a resolution of 3.27 Å (Fourier Shell Correlation = 0.143). Next, classification into eight classes without alignment with a focus mask around the A-, and P-sites of the large and small subunit yielded two classes with density in the A-site. The classes corresponded to one rotated (23.15% of all particles), and one non-rotated state (11.44% of all particles), respectively. Both states were extracted separately and refined using local refinement with increasing resolution limits to 5 Å followed by one round of CTF refinement without alignment. The rotated and the non-rotated states reached resolutions of 3.15 Å and 3.35 Å, respectively. Each class then was classified into five classes without alignment using a focus mask around the observed density in the A-site. Two classes obtained from the non-rotated state showed weak density in the A-site. The two classes were merged and classified further into eight classes. Two classes had A-site density of which one showed strong density corresponding to the hairpin in the A-site and tRNA Phe in the P-site. Particles for this class were extracted and aligned with increasing resolution limits to 5 Å. Finally, CTF refinement to 4 Å resolution without alignment and a step size of 50 Å was run and the final reconstructions were calculated using a beam-tilt corrected particle stack yielding final resolutions of 3.4 Å and 3.3 Å (Fourier Shell Correlation = 0.143).

The classification for the R conformation was done as described for the NR conformation. Classification into five classes yielded two classes with hairpin density. The classes were merged and classified into 8 classes of which four classes had weak density and one class yielded strong density. This class was extracted, CTF, and beam-tilt refined yielding a final resolution of 3.1 Å.

Finally, the obtained maps were sharpened in cisTEM and using the local resolution dependent function in phenix.autosharpen (Terwilliger et al., 2018).

Model building and refinement

As the starting model for refinement we used the structure of the E. coli 70S ribosome with a ternary complex (PDB ID 5UYL), omitting EF-Tu and the A-site tRNA. An NMR structure of the HIV-1 frameshifting element (PDB ID 1PJY) was used as the starting model for the hairpin and to generate secondary-structure restraints. Missing parts of the mRNA were built manually and the geometry was regularized in phenix.geometry_minimization before refinement. The A-site finger was modeled using nucleotides 873–904 from PDB ID 5KPS where the A-site finger is well-ordered. Protein secondary structure restraints were generated in Phenix (Adams et al., 2010) and edited manually. We generated base-pairing (hydrogen bonds) restraints using the ‘PDB to 3D Restraints’ web-server (http://rna.ucsc.edu/pdbrestraints/, [Laurberg et al., 2008]) and added stacking restraints manually for the hairpin, and A-site finger.

Initially, the ribosomal subunits, tRNA and the hairpin were separately fitted into the cryo-EM, using Chimera, followed by manual adjustments in Coot (version 0.9-pre) (Emsley et al., 2010). The structural model was refined using phenix.real_space_refine (Afonine et al., 2018) and alternated with manual adjustments in Coot. The final model was evaluated in MolProbity (Williams et al., 2018).