Biology subjects

Whitford, P.

Publications and source records attributed to Whitford, P..

4 recordsLinked to original sources

bioRxiv2026

Ribosomal Protein bL27 Protects Translating Ribosomes from tmRNA-SmpB

Bacterial ribosomal protein bL27 is universally conserved and its amino terminus is adjacent to the peptidyl transfer center, yet its role in translation remains unclear. Combining genetics, biochemistry and molecular dynamics, we show that bL27 has an unexpected role in preventing trans-translation, the bacterial ribosome rescue mechanism, from interfering with protein synthesis. Deletion of the bL27 gene causes a 10,000-fold decrease in viability and this defect is partially rescued by deletion of the gene encoding tmRNA, a critical molecule for trans-translation. Molecular dynamics simulations also indicate that bL27 can slow the movement of tmRNA on the ribosome. These data link trans-translation and bL27, and support a model in which the amino terminus of bL27 acts as a gatekeeper to prevent tmRNA from sterically interfering with tRNA on the ribosome.

biochemistry↗

bioRxiv2023

Structure of mitoribosome reveals mechanism of mRNA binding, tRNA interactions with L1 stalk, roles of cofactors and rRNA modifications

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 [A] resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transition in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide the most complete description so far of the structure and function of the human mitoribosome.

biochemistry↗

bioRxiv2021

Sterically-Confined Rearrangements of SARS-CoV-2 Spike Protein Control Cell Invasion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious, and transmission involves a series of processes that may be targeted by vaccines and therapeutics. During transmission, host cell invasion is controlled by a large-scale conformational change of the Spike protein. This conformational rearrangement leads to membrane fusion, which creates transmembrane pores through which the viral genome is passed to the host. During Spike-protein-mediated fusion, the fusion peptides must be released from the core of the protein and associate with the host membrane. Interestingly, the Spike protein possesses many post-translational modifications, in the form of branched glycans that flank the surface of the assembly. Despite the large number of glycosylation sites, until now, the specific role of glycans during cell invasion has been unclear. Here, we propose that glycosylation is needed to provide sufficient time for the fusion peptides to reach the host membrane, otherwise the viral particle would fail to enter the host. To understand this process, an all-atom model with simplified energetics was used to perform thousands of simulations in which the protein transitions between the prefusion and postfusion conformations. These simulations indicate that the steric composition of the glycans induces a pause during the Spike protein conformational change. We additionally show that this glycan-induced delay provides a critical opportunity for the fusion peptides to capture the host cell. This previously-unrecognized role of glycans reveals how the glycosylation state can regulate infectivity of this pervasive pathogen.

biophysics↗

bioRxiv2020

Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon-anticodon pairing

Modifications in the tRNA anticodon, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguaonosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop, immediately adjacent to the anticodon nucleotides 34-36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and m1G37 destabilize interactions of tRNAPro with the peptidyl site, causing large conformational changes typically only seen during EF-G mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome and the influence of slippery codons on the mRNA reading frame. IMPACT STATEMENTChemical modifications near the tRNA anticodon and specific mRNA-tRNA pairs combine to control the ribosomal three-nucleotide mRNA reading frame, essential for the sequential addition of amino acids into polypeptide chains. Data depositionCrystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB codes 6NTA, 6NSH, 6NUO, 6NWY, 6O3M, 6OSI)

biochemistry↗