Biology subjects

Zuber, P. K.

Publications and source records attributed to Zuber, P. K..

6 recordsLinked to original sources

bioRxiv2025

Molecular architecture and diversity of StopGo/2A translational recoding

Viral 2A sequences trigger a co-translational peptide bond formation "skipping" event, termed "StopGo", to generate two separate proteins from a single open reading frame without classical termination. To investigate the mechanism of StopGo, we determined the cryo-EM structure of a mammalian ribosome positioned at the foot-and-mouth disease virus 2A (F2A) site. The structure shows how interactions between the F2A nascent chain (NC) and the ribosomal exit tunnel induce a conformational change in the peptidyl transferase center that precludes further translation elongation but instead pre-exposes the P-tRNA:F2A-NC ester bond for hydrolysis and NC release. Additionally, we bioinformatically characterized variation and host association across nearly 10,000 StopGo sequences identified in virus genomes. We expanded the canonical core motif to (D/G/C/N)(V/I)ExNPGP and identified additional rare but functional variants. We also revealed several distinct upstream motifs that we showed biochemically to be important for StopGo activity. Interestingly, although StopGo is known to be functionally active in plants, we found no evidence for natural utilization of StopGo by plant viruses. Overall, these findings provide valuable insights into a unique translation recoding mechanism, and lay foundations for further optimization of multi-gene expression in biotechnology.

molecular biology↗

bioRxiv2025

Mechanism of translation initiation on endogenous eukaryotic circular RNAs

Eukaryotic circular RNAs (circRNAs) perform a wide variety of functions. A subset of circRNAs has been demonstrated to undergo translation in vivo. However, few insights exist on the underlying translation mechanisms. Here, we elucidate the basis of translation initiation in two naturally occurring circular RNAs, circMbl and circSfl. We show that in vitro prepared versions of both circRNAs are translated in eukaryotic cell lysates and cells. Initiation depends on the untranslated region (UTR) and can be reconstituted using only the 43S pre-initiation complex, eukaryotic initiation factors 4G, 4A, 4B, and, for circSfl, the RNA helicase DHX29. Functional assays and structural analysis of the initiation complexes suggest that scanning until recognition of the start codon follows initial ribosome landing occurring on AU-rich, accessible UTR patches upstream of the initiation sites. Together, these results provide key insights into how eukaryotic ribosomes engage with and initiate translation on circular endogenous transcripts.

molecular biology↗

bioRxiv2023

Concerted transformation of a hyper-paused transcription complex and its reinforcing protein

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an -helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a {beta}-barrel, which binds the ribosome. Our structures of ops-paused transcription elongation complexes alone and bound to the autoinhibited and activated RfaH reveal swiveled, pre-translocated pause states stabilized by an ops hairpin in the non-template DNA. Autoinhibited RfaH binds and twists the ops hairpin, expanding the RNA:DNA hybrid to 11 base pairs and triggering the KOW release. Once activated, RfaH hyper-stabilizes the pause, which thus requires anti-backtracking factors for escape. Our results suggest that the entire RfaH cycle is solely determined by the ops and RfaH sequences and provide insights into mechanisms of recruitment and metamorphosis of NusG homologs across all life. HIGHLIGHTS- The nontemplate DNA strand of an ops-paused transcription complex forms a hairpin - Autoinhibited RfaH binds and twists the ops hairpin to expand the RNA:DNA hybrid - RfaH-hairpin contacts are solely responsible for triggering RfaH activation - Upon recruitment, RfaH hyper-stabilizes the pause and promotes RNAP backtracking

biochemistry↗

bioRxiv2022

Structural and thermodynamic analyses of the β-to-α transformation in RfaH reveal principles of fold-switching proteins

The two-domain protein RfaH, a paralog of the universally conserved NusG/Spt5 transcription factors, is regulated by autoinhibition coupled to the reversible conformational switch of its 60-residue C-terminal KOW domain between an -hairpin and a {beta}-barrel. In contrast, NusG/Spt5-KOW domains only occur in the {beta}-barrel state. To understand the principles underlying the drastic fold switch in RfaH, we elucidated the thermodynamic stability and the structural dynamics of two RfaH- and four NusG/Spt5-KOW domains by combining biophysical and structural biology methods. We find that the RfaH-KOW {beta}-barrel is thermodynamically less stable than that of most NusG/Spt5-KOWs and we show that it is in equilibrium with a globally unfolded species, which, strikingly, contains two helical regions that prime the transition towards the -hairpin. Our results suggest that transiently structured elements in the unfolded conformation might drive the global folding transition in metamorphic proteins in general.

biophysics↗

bioRxiv2019

Escherichia coli NusG links the lead ribosome with the transcription elongation complex

It has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG.

molecular biology↗

bioRxiv2018

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH: ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

biochemistry↗