Why is transcription and translation simultaneously in prokaryotes

Yet, transcriptional and translational elongation rates can respond to independent signals, resulting in varying lengths of the nascent mRNA connecting the RNAP and the leading ribosome Conn et al. For instance, transcriptional pauses or backtracking reduce the distance between the leading ribosome and RNAP, which can give way to the formation of a collided expressome Kohler et al.

On the other hand, translational roadblocks will increase the distance between the leading ribosome and RNAP. As the mRNA connecting the two machineries in the coupled expressome is exposed to the solvent Wang et al. The in-cell architectures of Mycoplasma pneumoniae expressomes published recently show similarities to the E. On one hand, structures obtained from pseudouridimycin-halted M. On the other hand, M. It should be emphasized that all E.

Also, the questions of whether RNAP and the lead ribosome are physically coupled on the nascent mRNA in vivo and whether there is a mechanism to ensure or promote this coupling remain open. Where in the bacterial cell does CTT occur? The subcellular organization of the transcriptional and translational machineries offers clues to answer this question. RNAP spends most of its lifetime bound to DNA, either engaged in transcription or non-specifically searching promoters Endesfelder et al.

In rich media, RNAPs form nucleolus-like clusters engaged in rRNA transcription, but cluster assembly is independent of ongoing transcription Jin et al. Ribosome localization, on the other hand, differs considerably among species. In organisms with a high nucleocytoplasmic NC ratio Gray et al. This facilitates the encounter of the transcriptional and translational machineries, and the occurrence of CTT can be envisaged fairly intuitively in these organisms.

Yet, in many other species with low NC ratio, including E. The nucleoid-excluded localization of E. Hence, in these species, the transcriptional and translational machineries rarely encounter each other and the occurrence of CTT is less intuitive.

Several biophysical forces cause the subcellular nucleoid-vs-ribosome segregation. By avoiding extensive contacts with the inner membrane, the DNA polymer maximizes its number of available conformational states, i. This segregation is further accentuated by volume exclusion forces mutually exerted by the nucleoid polymer against the bulky polysomes Mondal et al.

Lastly, electrostatic repulsion forces between negatively charged nucleic acids, in this case, chromosomal DNA and RNA-rich ribosomes Joyeux, , and phase separation effects Joyeux, could also account for the observed antilocalization of nucleoids and ribosomes.

Indeed, it was shown that highly transcribed gene loci migrate to the nucleoid periphery Stracy et al. The association of several RNAPs into biomolecular condensates at highly transcribed loci Ladouceur et al. Supporting this hypothesis, the cotranscriptional association of bulky ribosomes amplifies loci migration to the nucleoid periphery Yang et al. CTT scenarios in low nucleocytoplasmic NC ratio species. B Via transertion, gene loci encoding membrane proteins emerge from the nucleoid to the inner membrane, where CTT readily occurs.

C Intranucleoidal translation initiation. Free ribosomal subunits penetrate the nucleoid and a 30S subunit associates with the SD sequence of the nascent transcript C-i. A 50S subunit associates with the pre-initiation complex, forming a 70S ribosome C-ii. D Uncoupled transcription-translation UTT.

Transcription takes place within the nucleoid translation-independently D-i , and ribosome-free transcripts navigate the cell to their corresponding destination in the cytoplasm or in the membrane D-ii , where they are locally translated D-iii. Gene loci encoding membrane proteins are thought to be expressed by coupled transcription-translation-membrane insertion, a mechanism known as transertion Woldringh, This implies that certain gene loci migrate from the nucleoid mesh to the vicinity of the inner membrane and become exposed to ribosome-rich regions, where CTT could readily occur Figure 4B.

Although the occurrence of transertion still awaits direct experimental validation Roggiani and Goulian, , the notion is supported by the demonstration that a small population of ribosomes and RNAPs resides in the membrane vicinity Herskovits and Bibi, ; Bakshi et al.

Alternatively, it was shown in E. This implies that canonical translation initiation, which is conducted by a free 30S subunit that recognizes the Shine-Dalgarno SD sequence of the nascent transcript, could initiate within the nucleoid in a cotranscriptional manner. An additional possibility is that transcription and translation are not compulsorily coupled. In this scenario, transcription takes place within the nucleoid, in spatiotemporal separation from translation, and the mRNAs then navigate the cytoplasm to their final destination where they are locally translated Kannaiah and Amster-Choder, ; Irastortza-Olaziregi and Amster-Choder, ; Figure 4D.

Coupled transcription-translation is widely accepted by the microbiology community and supported by extensive work. Yet, most knowledge regarding CTT emanates either from in vitro studies or from experiments conducted with only a handful of genes.

Importantly, the subcellular segregation of the transcriptional and translational machineries observed in some species raises the possibility that these processes could take place in spatiotemporal separation see section The Cell Biology of CTT.

Indeed, several lines of evidence have recently challenged the classical view that transcription is inherently coupled to translation, and the global occurrence of CTT has been questioned.

The Fredrick lab developed a hammerhead ribozyme-based reporter system that enables measuring and comparing protein synthesis carried out by limited vs. They applied this system to study the translation of six adjacent gene-pairs that are cotranscribed in the same operon. In a tight CTT scenario, limiting translation rounds should bring the relative protein amounts for each gene-pair close to , as protein synthesis of the two co-transcribed genes would presumably be carried out by a leading ribosome physically coupled to RNAP.

However, for five out of six gene-pairs, they observed that the relative protein synthesis derived from limited translation rounds of the two genes was not close to Rather, the ratio was similar to that measured for unlimited translation rounds. This indicates that the first translational rounds occur independently of transcription, i. Importantly, when these experiments were repeated with an RNAP mutant showing reduced transcription elongation rate, which, presumably, facilitates the physical coupling of the leading ribosome with RNAP see section The Molecular Architecture of CTT , protein production after limited translation rounds was closer to for one of the tested gene-pairs Chen and Fredrick, These results support a model where the physical coupling of transcription and translation is a stochastic event, which depends on the rates of transcription and translation elongation.

These observations indicate that physical association between the leading ribosomes and RNAPs, as well as coordinated elongation by the two machineries, is significantly less common than currently assumed, implying that RNAP often transcribes without a linked ribosome Chen and Fredrick, This indicates that, in the absence of transcription-halting antibiotics, the majority of those ribosomes are engaged in CTT, but that they do not translate in physical association to RNAP.

Similar conclusions emanated from studying the relationship between termination efficiency TE at intrinsic terminators and their intragenic position Li et al. This position-dependent loss of TE was explained by the fact that ribosomes follow and catch up RNAPs closely enough to prevent the formation of terminators Li et al.

Indirectly, the dependence of TE on the distance from the start codon implies that transcription of the 5'-proximal coding sequences occurs translation-independently see section The Molecular Architecture of CTT. Yet, further evidence supports the idea that transcription and translation in some cases are completely uncoupled. It is accepted that translation increases mRNA stability by ribosome shielding against ribonucleolytic attack Iost and Dreyfus, ; Makarova et al. A reassessment of RNA decay patterns unraveled that, contrary to the widespread idea that RNA decay is exponential, two-thirds of the analyzed transcripts followed a biphasic degradation pattern, with a very steep decay at short post-transcription times followed by an exponential decay at longer times Deneke et al.

These results suggest that most transcripts spend a minor fraction of their lifetime in a ribosome-free form, where they are highly vulnerable to ribonucleases until ribosomes engage in translation and slowdown mRNA decay. At the same time, similar to what happens in eukaryotic cells, other RNA-binding proteins could be responsible for the protection of the transcripts till they are translated.

Whatever the reason is, these results imply that transcription is not tightly coupled to translation for most genes Deneke et al. Furthermore, previous work from our lab and others showed that transcripts can localize to sites overlapping with the localization of their encoded proteins in the cytoplasm, the membrane, or the poles of E. Very importantly, these localization patterns were preserved when the translation of the tracked transcripts was inhibited by antibiotics, translational roadblocks, or mutations Nevo-Dinur et al.

Similarly, a transcript encoding a short membrane protein localized to the membrane even when the SD sequence of the mRNA was deleted Steinberg et al. Likewise, translation-independent RNA localization has been observed in cyanobacteria, where transcripts encoding photosystem components localized to thylakoid membranes in the presence of puromycin concentrations that inhibit translation and detach ribosomes from mRNAs Mahbub et al.

These observations imply that bacterial mRNAs can skip tight CTT, navigate the cytosol as ribosome-free transcripts, and undergo local translation once they reach their corresponding destination Nevo-Dinur et al.

Another recent publication from our lab, which reports the distribution of E. Collectively, this evidence supports the notion that CTT is not as predominant as currently assumed, and that spatiotemporally uncoupled transcription-translation UTT could be responsible for substantial gene expression. For E. Then again, early evidence already indicated that transcription can be tuned independently of translation. Although mRNA transcription-translation rates change and equalize each other at different growth rates, rRNA transcription rates also vary according to growth rates Vogel and Jensen, b , suggesting that ribosome-independent mechanisms exist in bacteria for determining transcription elongation rates.

As discussed in section Coordination of CTT, the application of sublethal concentrations of fusidic acid, which slows down ribosome translocation, did not affect RNAP elongation rates Zhu et al. Besides, when ribosomes stalled at proline-rich sequences of E. In further agreement with ribosome-independent transcription, when backtracked TECs are pushed and reactivated by the leading ribosome, transcription elongation restarts even when translation elongation is inhibited Stevenson-Jones et al.

Likewise, in M. How can the transcription processivity of TECs that are engaged in mRNA transcription be maintained in the absence of a coupled ribosome? The trafficking of transcription elongation and termination factors can offer a partial explanation for this.

NusG recruitment to E. Thus, translation-independent TECs could show less affinity for NusG and, consequently, may be less prone to Rho-mediated termination. Effects related to DNA supercoiling offer alternative explanations for this transcriptional cooperation.

A mathematical model predicts that the torque created by transcription elongation over the DNA double helix pushes and pulls TECs located in close proximity forward without the mediation of any physical contact among each other Heberling et al.

Recently, a publication from the Jacobs-Wagner lab evidenced that, as long as gene promoters remain induced and multiple RNAPs initiate transcription of the lacZ gene, elongation rates of ongoing transcription are maintained by the mutual cancelation of positive and negative DNA supercoiling upstream and downstream the TEC convoy Kim et al.

Regardless of the actual underlying mechanism, we suggest that the cooperation between RNAPs could suffice for maintaining the transcription processivity required for UTT. In vitro studies showed that the B. Although, as reported for E. Accordingly, transcription terminators in B. A bioinformatic exploration using this short distance between stop codons and intrinsic terminators as a proxy for runaway transcription suggested that this mode of gene expression could be a fairly widespread phenomenon in bacteria Johnson et al.

In other words, transcription may occur translation-independently, and mRNAs could be transcribed and translated in spatiotemporal separation. Dynamics of transcription elongation over 5'-proximal coding sequences are of special interest for UTT. Furthermore, transcription of 5'-proximal coding sequences is anyhow expected to occur translation-independently see section The Molecular Architecture of CTT , so why cannot this type of regulation continue to be exerted over the downstream ribosome-naked transcript before translation begins?

Below, we describe several molecular factors and mechanisms that, when acting in cooperation, potentially promote UTT in a way that the three challenges mentioned above would be satisfactorily resolved Figure 5. Evidence supporting the capacity of these factors and mechanisms to act cotranscriptionally on nascent mRNAs is presented. Mechanisms potentially enabling UTT. Cotranscriptional events, such as association with an RBP or with an sRNA, as well as riboswitch formation, prevent transcription termination by Rho and association with the leading ribosome.

When ribosome-free transcripts are released to the cytoplasm, these transcript-protecting events counteract the activity of ribonucleases. Of note, although drawn linearly, transcripts supposedly acquire complex secondary and tertiary structures that confer further protection. This protection also prevents mRNA translation until they reach their final destination.

RBPs have gained major interest as a consequence of their capability to regulate transcript fate by affecting mRNA translation and stability Holmqvist and Vogel, ; Richards and Belasco, , and emerging evidence suggest they can promote UTT.

For instance, Synechocystis RBPs Rbp2 and Rbp3 are required for the translation-independent thylakoid membrane localization of transcripts encoding photosystem proteins Mahbub et al.

Likewise, Grad-seq experiments performed in Salmonella and E. Considering their transcript fate-determining activities, these RBPs emerge as potential candidates for promoting UTT. Cold Shock Proteins CSPs belong to an evolutionarily widespread family of small, acidic proteins that were originally discovered as mediators of cold shock response, but it is currently understood that their function exceeds adaptation to cold reviewed in Ermolenko and Makhatadze, ; Horn et al.

Notably, the presence of at least one csp gene in the cell is essential for viability in B. The E. Bioinformatical explorations unraveled a sequence-level bias towards U-richness in transcripts encoding integral membrane proteins in E. Information published by our lab confirmed that the U-richness in membrane-traversing domains is important for their membrane localization, as predicted bioinformatically Kannaiah et al.

Interestingly, E. Moreover, CspE overexpression causes the accumulation of ribosome-free transcripts encoding membrane proteins in the cytoplasm and positively affects their translation in the membrane Benhalevy et al. This suggests that, via CspE mediation, these transcripts avoid CTT until they reach the membrane, where they are locally translated. Whereas CspE levels remain constant through different growth phases, CspC levels increase upon entry into stationary phase, which results in the stabilization of rpoS transcripts Shenhar et al.

These transcript-stabilizing capabilities can be explained by the tendency of CspE to bind poly A sequences, counteracting the ribonucleolytic activity of PNPase and RNase E Feng et al. Considering that most transcripts undergo polyadenylation Mohanty and Kushner, , CspE could act as a global molecular shield against the concerted poly A -dependent exoribonucleolytic action of PNPase in the 3'-end and the endonucleolytic attack of RNase E in internal cleavage sites of nascent transcripts.

Indeed, CspE binds nascent RNAs and acts as an antiterminator by melting secondary structures of intrinsic terminators Hanna and Liu, ; Bae et al. Collectively, these pieces of evidence indicate that CSPs can promote the uncoupling of transcription and translation by counteracting RNase activity over transcripts and promoting translation-independent transcription over intrinsic terminators.

Cold shock proteins show UTT-promoting activities in other species. Similarly, CspE binds and stabilizes yciF transcripts, conferring Salmonella increased resistance to bile salts by impermeabilizing the cell membrane Ray et al.

Also, in B. Their function resembles that of FRGY2, a CSD-containing protein that binds mRNAs in the nucleous of frog oocytes and protects transcripts from degradation and translation in the cytoplasm until they are released in a regulated manner during oocyte development Bouvet and Wolffe, Their multifaceted functions as transcription antiterminators and antiribonucleolytic shields make CSPs promising subjects for future study regarding their putative role as UTT facilitators.

In addition to multiple interactions with rRNAs within the ribosome, ribosomal proteins RPs perform extraribosomal moonlighting functions as free RBPs, often involved in gene regulation reviewed in Bhavsar et al. One example is the S1 RP in E.

In line with these results, depletion of S1 leads to the destabilization of these transcripts in vivo Briani et al. Whether S1 counteracts PNPase in vivo by promoting CspE-dependent transcript protection see above or through an alternative mechanism remains unknown.

Additionally, S1 was shown to increase protein secretion that is mediated by the hemolysin signal peptide, and this was accompanied by the stabilization of the corresponding transcripts Khosa et al.

Thus, it is plausible that S1 binds nascent transcripts cotranscriptionally and protects them from ribonucleolytic attack, thus favoring the occurrence of UTT. This autoregulation also represses the translation of several other RPs cotranscribed within the same operon. To exert such CTT disruption, S4 would need to act cotranscriptionally. L4 binds the regulatory CTD of RNase E and inhibits its activity, leading to stabilization of a subset of stress-related transcripts crucial for cell survival Singh et al.

Among the L4-stabilized transcripts is that of the triptophanase tna operon, which is subjected to an additional layer of regulation by L4 that is degradosome-independent. Specifically, L4 overexpression increases the stability of the tnaCAB transcript but causes translational repression of the TnaA protein by binding to the spacer between tnaC-tnaA Singh et al. This could lead to the partial disruption of CTT in this operon, i. Interestingly, this translational repression of tnaA takes place at early stationary phase to prevent the degradation of tryptophan, which is required for long-term survival through deep stationary phase Singh et al.

Importantly, L4 binds its cognate transcript to attenuate its transcription Lindahl et al. Thus, L4 could act cotranscriptionally as an antitranslation agent also over other nascent transcripts. Further investigation of extraribosomal functions of RPs should lead to a better understanding of these activities that are potentially involved in UTT. In all these cases Hfq binding leads to translational repression of the target transcripts. These activities can be explained by the observation that in enterohemorrhagic E.

Likewise, translational repression by Hfq plays a central role in the catabolic repression of the opportunistic pathogen Pseudomonas aeruginosa.

Hfq was also shown to preferentially bind intrinsic terminator sequences in Salmonella Holmqvist et al. Collectively, this evidence indicates that Hfq binds intrinsic termination sites in order to promote polyadenylation and, by binding to these poly A sequences, protects 3'-UTRs and upstream sequences from ribonucleolytic decay.

Furthermore, Hfq shares topological similarities with YaeO, the only so-far discovered protein that binds and inhibits Rho in E. Accordingly, Hfq suppresses Rho-dependent termination by simultaneously binding Rho and AU-rich sequences located upstream rut sites Rabhi et al. Recently, it has been shown that Hfq pervasively binds nascent transcripts in E.

The helix-like localization of Hfq observed in E. Therefore, Hfq emerges as a potential UTT-promoting factor by antiterminating ribosome-free transcription that could otherwise be terminated by Rho.

The fact that Hfq can interact with nascent transcripts implies that the antitranslation and antiribonuclease roles of Hfq described here could come into play cotranscriptionally to disrupt CTT and protect nascent mRNAs from RNases. Although initially discovered as a regulator of glycogen biosynthesis upon entry into stationary phase Romeo et al.

CsrA has been shown to bind hundreds of transcripts in E. The main regulatory activity of CsrA is via translational repression Dugar et al.

Similarly, CsrA binds at two sites in the 5'-UTR, including the SD sequence, of transcripts encoding the transcriptional regulator NhaR, which responses to high sodium concentrations and alkaline pH Pannuri et al. In enteropathogenic E. After releasing the effector, free CesT binds and inhibits CsrA, leading to derepression of NleA and its subsequent translation and translocation to the host cell Katsowich et al.

In all examples discussed here, translation inhibition was not accompanied by decreased transcript stability. Consequently, this can lead to the accumulation of ribosome-free transcripts in the cytoplasm, resembling the phenomenon observed upon CspE and S1 overexpression see above. For instance, CsrA was shown to be essential for E. CsrA also displays transcription-related activities, e.

Furthermore, in P. Furthermore, about a third of ProQ binding events take place in sites overlapping with RNase E cleavage sites Chao et al. Thus, it is plausible that ProQ accesses and binds nascent transcripts cotranscriptionally, promoting UTT by protecting untranslated transcripts against ribonucleolytic decay. Yet, NusA could possibly counteract Rho-dependent termination in certain instances.

Specifically, NusA mutants with increased affinity for binding NusA utilization nut sites decreased Rho-dependent termination at specific cases where nut and rut sites overlap Qayyum et al. Thus, regarding UTT, NusA could facilitate translation-independent transcription of mRNAs by interfering with certain Rho-dependent termination events on nascent ribosome-free transcripts. Other Nus factors are involved in processive antitermination PA mechanisms.

Opposite to dedicated antiterminators, PA factors associate with and modify TECs to promote transcriptional readthrough over multiple transcription terminators distally located in the operons under their regulation Goodson and Winkler, For example, the NusG paralogue LoaP regulates transcription through termination sites located within two antibiotic biosynthesis operons in Firmicutes , Actinobacteria , and Spirochaetes Goodson et al.

Deletion of loaP led to a reduction in transcript levels of LoaP regulons. LoaP is thought to processively antiterminate intrinsic terminators, an activity that requires the 5' leader sequence of the transcripts under its regulation Goodson et al.

All in all, the PA activity of such Nus paralogs on their specific target operons could favor UTT by alleviating the need for a translationally coupled ribosome for counteracting intrinsic and Rho-dependent terminators. Riboswitches are regulatory elements located in the 5'-UTR of mRNAs that are comprised of two modules: a structurally complex aptamer that binds a ligand and an expression platform that is regulated by the aptamer structural folding.

The coupling could allow translation to prevent transcriptional pausing, backtracking, and termination. Simultaneous Gene Transcription and Translation in Bacteria. In bacteria , mRNA is translated into protein as soon as it is transcribed. Unlike eukaryotic cells, bacteria do not have a distinct nucleus that separates DNA from ribosomes, so there is no barrier to immediate translation. There is no such structure seen in prokaryotes.

Another main difference between the two is that transcription and translation occurs simultaneously in prokaryotes and in eukaryotes the RNA is first transcribed in the nucleus and then translated in the cytoplasm.

Mostly, yes and this is somehow meant to be. There is an enourmous amount of alternative splicing in human cells, meaning that a gene can encode various different transcripts depending on their splice condition.

Why does transcription occur in the nucleus and not in the cytoplasm in eukaryotes? DNA is always inside the nucleus unless the cell is dividing. RNA splicing occurs in eukaryotes but generally not in prokaryotes.

In eukaryotes organisms with a nuclear membrane , DNA undergoes replication and transcription in the nucleus, and proteins are made in the cytoplasm. RNA must therefore travel across the nuclear membrane before it undergoes translation. This means that transcription and translation are physically separated. For prokaryotes DNA replication, transcription, and translation occur inside of the cytoplasm. For eukaryotes translation occurs inside of the cytoplasm.

For a protein-coding gene, the RNA copy, or transcript, carries the information needed to build a polypeptide protein or protein subunit. Eukaryotic transcripts need to go through some processing steps before translation into proteins. Prokaryotic transcription occurs in the cytoplasm alongside translation.

Prokaryotic transcription and translation can occur simultaneously. This is impossible in eukaryotes, where transcription occurs in a membrane-bound nucleus while translation occurs outside the nucleus in the cytoplasm. In prokaryotes genetic material is not enclosed in a membrane-enclosed nucleus and has access to ribosomes in the cytoplasm. Transcription is controlled by a variety of regulators in prokaryotes. Thus, in eukaryotes tanscription and translation occur in different cellular compartments that are separated by a membrane barrier such as transcription occurs inside the nucleus and translation occurs in the cytoplasm.

In prokaryotes, protein synthesis which is called translation starts while the mRNA is still being synthesized which is called transcription. This is because there is no nucleus in prokaryotes that separates the transcription and translation process. Therefore, when bacterial genes are transcribed then transcripts begin to translate immediately. Prokaryotic transcription occurs in the cytoplasm alongside translation and both processes occur simultaneously.