Molecular basis of mRNA delivery to the bacterial ribosome

Abstract

Protein synthesis begins with the formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through base pairing with the Shine Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S subunit. Here we examined ribosome recruitment to nascent mRNAs using cryo-EM, single-molecule fluorescence co-localization, and in-cell crosslinking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S subunit activation. Additionally, bS1 mediates the stimulation of translation initiation by RNAP. Together, our work provides a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.

Figure 1:. Cryo-EM reconstructions of translation initiation complexes linked to transcription.

(A) Consensus reconstruction of mRNA delivery complex particles (gold, composite map; white, map filtered to 10 Å) revealed density surrounding the 30S platform, mRNA exit channel, and bS1 (crosshair). This was identified to be a TEC associated flexibly with 30S in a reconstruction (green, RNAP_dlv) obtained by partial signal subtraction, focused classification and refinement. (B) Reconstruction of a 30S-PIC with accommodated mRNA in the ribosomal P-site and bound to fMet-tRNA^fMet (purple). (C) Reconstruction of NusG-coupled RNAP_exp-30S complex in which mRNA (pink) enters the ribosomal decoding center through the mRNA entry channel.

Figure 2:. mRNA delivery involves SD-aSD duplex inversion, bS1 arch formation and RNAP_dlv association.

(A) Structural model and cryo-EM map (left) and schematic (right) of the 30S-PIC platform region showing the SD-aSD duplex orientation upon mRNA accommodation. (B) Structural model and cryo-EM map (left) and schematic (right) of mRNA delivery complex (30S_dlv) 30S platform region showing the SD-aSD duplex orientation during mRNA delivery. Relative to the 30S-PIC, the aSD is rotated close to 16S residue 1533 (curved black arrow) to allow the mRNA to anneal in an inverted orientation. The mRNA downstream of the SD contacts bS1-OB2. (C) Cryo-EM reconstruction (top; cyan, map filtered to 5 Å; white, map filtered to 10 Å) and structural model (bottom) of a 30S complex in which mRNA is delivered through the bS1 arch. (D) The RNA binding surface of the bS1 arch faces the 30S to form a channel for the delivered mRNA. Segmented reconstruction filtered to 5 Å (left, two contour levels) and structural model indicating representative mRNA path (right, dashed pink line). The mRNA binds bS1-OB2 to bS1-OB4 and connects bS1-OB2 and the aSD sequence at the 16S rRNA 3′-end. bS1 is anchored to ribosomal protein uS2 through its N-terminal helix and bS1-OB1. bS1-OB3 and bS1-OB4 interact with ribosomal protein bS6. (E) The bS1 arch adopts an alternative compact conformation (dark blue) in addition to the extended conformation (cyan) shown in panels C and D. (F) In mRNA delivery complexes, the RNAP_dlv orientation directs nascent mRNA towards the 30S. TEC focused cryo-EM reconstruction (left) at full resolution (colored) and filtered to 8 Å (white overlay) and structural model (right).

Figure 3:. 30S activation regulation by bS1 and SD-aSD position.

(A) Two inactive 30S conformations were identified by classification of NusG-coupled 30S-RNAP_exp particles. In both, RNAP is coupled to 30S by NusG and delivers the mRNA to the mRNA entry channel as observed in transcribing-translating expressomes. Cryo-EM reconstruction (left, gold and red composite map; orange and cyan, h44 and bS1 map filtered to 6 Å and overlaid) and structural model (right) of inactive state 1 shows h44 in the mRNA exit channel interacts with ribosomal protein bS1. Consequently, the SD base-pairs with the aSD in the ribosomal A-site and prevents correct folding of the decoding center. (B) In inactive state 2, h44 is accommodated on the 30S subunit interface side but the decoding center has not correctly folded. mRNA delivery by RNAP_exp produced SD-aSD base-paring in the ribosomal A-site, hindering activation as in inactive state 1. (C) The positions of bS1 and h44 in inactive state 1 suggest bS1-OB2 contacts non-canonical Watson-Crick base pairs of h44. (D) The position of the SD-aSD helix in inactive states (left, only inactive state 2 shown) overlaps with an accommodated tRNA bound to the A-site codon in a translation elongation complex (right).

Figure 4:. 30S recruitment to mRNA is promoted by bS1 and RNAP and in-cell crosslinking confirms RNAP_dlv position.

(A) Schematic of SiM-KARB experiment showing immobilization of pTEC-38 (left) or RNA-38 (right) and TIRF measurement of 30S binding. (B) Association (k_on) and dissociation (k_off) rate constants calculated from hidden Markov models for 30S binding to pTEC-38 (blue) and RNA-38 (purple), in the presence (+) or absence (−) of bS1. Values of k_on and k_off are reported in table S3. The total number of molecules analyzed were N_(pTEC+bS1) = 180, N_{(pTEC-∆bS1)} = 197, N_(RNA-38+bS1) = 130, N_(RNA-38-bS1) = 152. (***P < 0.01, **P < 0.025, *P < 0.05). (C) In-cell CLMS interaction map of RNAP and ribosomal proteins. Crosslinks between NusG and uS10 (red line) are consistent with NusG-coupled 30S and expressome models. Crosslinks between β-bS1, β-uS15 and β′-uS15 (green lines) are consistent with mRNA delivery complex models. Line thickness indicates number of crosslinks supporting each interaction (thin lines, single crosslink; medium lines, two crosslinks; thick lines, more than two crosslinks). (D) RNAP-30S subunit CLMS flexibility analysis. Accessible interaction space analysis showing the volume occupied by the RNAP center of mass (CoM) consistent with at least two CLMS restraints performed with DisVis (45) Structural models of RNAP_exp-ribosome complexes are consistent with one identified region (red density), and models of RNAP_dlv-30S complexes are consistent with the other identified region (green density).

Figure 5:. Model for 30S recruitment to the transcription elongation complex and establishment of coupling between RNAP and the ribosome.

Free 30S is pre-dominantly inactive in vivo, which is characterized by h44 (orange) occupying the mRNA exit channel and interacting with bS1 (cyan, left, both pathways) or h44 not folding correctly on the subunit interface side (not shown for clarity). The 16S rRNA 3′-end occupies the main mRNA binding channel (aSD highlighted in green, left). A TEC (grey, left, middle) may encounter a 30S so bS1 binds the nascent mRNA and guides the SD (purple) to the aSD (pathway I, right). Initiation factors (IF1, IF2, and IF3) and fMet-tRNA^fMet will bind and form a bona-fide 30S PIC with an accommodated mRNA and this may help RNAP to occupy the expressome position (RNAP_exp, middle, accommodated state). Alternatively, RNAP may bind an inactive 30S subunit in the expressome position but fails to activate the small subunit (pathway II, right). Initiation factors may allow full activation so both pathways could lead to formation of a transcribing-translating expressome (middle, right).