Two Old Dogs, One New Trick: A Review of RNA Polymerase and Ribosome Interactions during Transcription-Translation Coupling

Abstract

The coupling of transcription and translation is more than mere translation of an mRNA that is still being transcribed. The discovery of physical interactions between RNA polymerase and ribosomes has spurred renewed interest into this long-standing paradigm of bacterial molecular biology. Here, we provide a concise presentation of recent insights gained from super-resolution microscopy, biochemical, and structural work, including cryo-EM studies. Based on the presented data, we put forward a dynamic model for the interaction between RNA polymerase and ribosomes, in which the interactions are repeatedly formed and broken. Furthermore, we propose that long intervening nascent RNA will loop out and away during the forming the interactions between the RNA polymerase and ribosomes. By comparing the effect of the direct interactions between RNA polymerase and ribosomes with those that transcription factors NusG and RfaH mediate, we submit that two distinct modes of coupling exist: Factor-free and factor-mediated coupling. Finally, we provide a possible framework for transcription-translation coupling and elude to some open questions in the field.

Keywords: NusG; RNAP; RfaH; bacteria; coupling; nascent RNA; ribosomal subunits; ribosome; transcription; translation.

Figure 1

Schematic representation of transcription polarity, premature transcription termination on long intervening nascent RNA, and synchronization of transcription and translation rates. (a) Transcription polarity is caused by a premature stop codon (STOP sign) on the nascent RNA (nRNA, in red). Translation of the nascent RNA will terminate and the ribosome (in yellow and blue, for small and large ribosomal subunits, respectively) will prematurely dissociate from the nascent RNA. This allows the Rho transcription termination factor (in purple) to reach the RNA polymerase (RNAP, in green) and induce premature transcription termination. (b) A long intervening nascent RNA allows the Rho factor to bind ahead of the ribosome or allow the intrinsic transcription terminator to fold (hairpin structure indicated with a red capital T). In both instances, transcription terminates. (c) Synchronization of transcription rate to translation rate. The running ahead of the RNAP will cause the polymerase to pause and backtrack (complex on the left). The translating ribosome will push the RNAP forward and reactivate its transcription activity (complex on the right). This running ahead and pausing to wait for the ribosome synchronizes the transcription rate to the translation rate.

Figure 2

Schematic representation of the coupling of transcription and translation on highly expressed genes under fast growing conditions. RNA polymerase (RNAP, in green) initiates transcription on the DNA (in brown) within the nucleoid (brown shaded area). As soon as the polymerase has transcribed a sufficiently long nascent RNA (in red), translation will ensue (large and small ribosomal subunits in blue and yellow, respectively). During coupling, the active gene is relocalized to the interface of the nucleoid and cytoplasm. The progression of this relocalization is indicated by arrows and by the progressive increase in opacity of the DNA, RNAP, nascent RNA, and ribosomal subunits.

Figure 3

Display of the RNAP-ribosome interactions and contact points identified by biochemical and cryo-EM studies. (a) Ribosomal proteins (in orange) that influence the RNAP activity by themselves (bS1 [65], uS4 [60], and uS10 [63]) are mapped onto the small ribosomal subunit (30S), derived from the cryo-EM structure of the small ribosomal subunit bound to the RNAP (30S•RNAP) [71]. Because ribosomal protein bS1 is only partially resolved in this structure, we outlined the approximate position of the remaining protein (orange shaded area). In addition, the mRNA entry (blue circle) and exit (red circle with dashed black border indicating its positions behind bS1) sites on the small ribosomal subunit are indicated. In the right corner is a cartoon representation of the direction of the view displayed of the small ribosomal subunit. (b) Ribosomal proteins and RNA helices contacting the RNAP upon binding of the small ribosomal subunit to RNAP. Shown are the proteins identified to be close to the RNAP in the cryo-EM structure of 30S•RNAP in Demo et al. [71] and by chemical crosslinking in Fan et al. [73]. (Proteins observed only in Demo et al. are in yellow, those shared by Demo et al. and Fan et al. are in orange, and those observed only in Fan et al. are in red.) (c) Ribosomal proteins (in orange) contacting the RNAP in the cryo-EM structure of a ribosome translating a nascent RNA as it is being synthesized, also known as expressome [72]. Interactions between the C-terminal domain of one of the two α subunits of the RNAP with the ribosome were omitted for clarity. (d,e) Contact interfaces between the RNAP and the small ribosomal subunit as seen in the cyro-EM structures of the 30S•RNAP complex (d) and the expressome (e). In both representations, the view is onto the contact areas (gray shaded areas) on the RNAP (green) and on the small ribosomal subunit (yellow). Also indicated is the β flap-tip of the RNAP (red, marked with FT), past which the nascent RNA exits the RNAP to enter the small ribosomal subunit.

Figure 4

The effect of the tethering of RNAP and ribosomes by nascent RNA on the RNAP•ribosome complex formation. (a) Dependence of the RNAP•ribosome complex formation on the length of the intervening nascent RNA. The intervening nascent RNA was modeled as a freely jointed chain. The local concentration of the first trailing ribosome around the RNAP that it is tethered to (left y-axis) and the fraction of the RNAP-ribosome complex formation (in blue, right y-axis) are plotted against the length of the intervening nascent RNA. Local concentration and the fraction of complex formation were calculated, following Conant et al. [79] and Rippe [84]. (b) Schematic representation of the binding equilibrium dynamics between the first trailing ribosome (in blue and yellow for large and small ribosomal subunits, respectively) and the RNAP (in green), tethered via the nascent RNA (red). Binding of the RNAP and the ribosome will cause the intervening nascent RNA to loop out and away from the RNAP-ribosome complex.

Figure 5

Model of the RNAP-ribosome arrangements during the factor-free and factor-mediated coupling of transcription and translation. The representation of the small ribosomal subunit (30S in yellow) is the same in all panels, with both the RNAP binding sites facing the reader. The RNAP, the large ribosomal subunit (50S), DNA, and the nascent RNA are shown in green, blue, brown, and red, respectively. NusG and RfaH, the factors that physically link the RNAP and the ribosomes during factor-mediated coupling, are shown in dark red. (a) Co-localization of the RNAP and the small ribosomal subunits within the nucleoid. (b) Recruitment of the nascent RNA to the small ribosomal subunit during the first step of translation initiation. Also shown is the positioning of the 5’ end of the nascent RNA, relative to the 3’ end of the ribosomal RNA of the small ribosomal subunit (3’ end rRNA). In many cases, both ends engage in base pairing interactions. (c) During translation initiation, the RNAP relocalizes on the 30S subunit from the mRNA exit site shown in (a) and (b) to the mRNA entry site. Shown is the RNAP-ribosome complex with the shortest intervening nascent RNA. (d) Recruitment of the transcription factor RfaH to the RNAP, which has transcribed and paused at the ops signal sequence. The RfaH’s C-terminal domain undergoes a conformational change from an all α helical to an all β sheet structure. (e) Recruitment of the small ribosomal subunit (30S) to the RNAP-RfaH complex before initiation of translation. (f) During factor-mediated coupling, the RNAP and the ribosome are held close to each other by either the transcription factor RfaH or NusG. The NusG-mediated coupling is established by the binding of NusG to the factor-free coupled RNAP and ribosome.