David S. McPheeters

Autogenous regulatory site on the bacteriophage T4 gene 32 messenger RNA.

We have identified the binding site on the bacteriophage T4 gene 32 mRNA responsible for autogenous translational regulation. We demonstrate that this site is largely unstructured and overlaps the initiation codon of gene 32 as previously predicted. Co-operative binding of gene 32 protein to this site specifically blocks the formation of 30 S-tRNA(fMet)-gene 32 mRNA ternary complexes and initiation of translation. The translational operator is bound co-operatively by gene 32 protein and this binding is facilitated by a nucleation site far upstream from the initiation codon. A similar unstructured mRNA lacking this nucleation site is also bound co-operatively, but only at concentrations of gene 32 protein higher than those needed to repress binding of ribosomes to the gene 32 mRNA. Some sequence-specific interactions may also influence this binding. Comparison of the bacteriophage T2, T4 and T6 gene 32 operator sequences leads us to propose that the nucleation site is a pseudoknot.

Spatial Organization of Protein-RNA Interactions in the Branch Site-3′ Splice Site Region during pre-mRNA Splicing in Yeast

ABSTRACT A series of efficiently spliced pre-mRNA substrates containing single 4-thiouridine residues were used to monitor RNA-protein interactions involving the branch site-3′ splice site-3′ exon region during yeast pre-mRNA splicing through cross-linking analysis. Prior to the assembly of the prespliceosome, Mud2p and the branch point bridging protein cross-link to a portion of this region in an ATP-independent fashion. Assembly of the prespliceosome leads to extensive cross-linking of the U2-associated protein Hsh155p to this region. Following the first step of splicing and in a manner independent of Prp16p, the U5 small nuclear ribonucleoprotein particle-associated protein Prp8p also associates extensively with the branch site-3′ splice site-3′ exon region. The subsequent cross-linking of Prp16p to the lariat intermediate is restricted to the 3′ splice site and the adjacent 3′ exon sequence. Using modified substrates to either mutationally or chemically block the second step, we found that the association of Prp22p with the lariat intermediate represents an authentic transient intermediate and appears to be restricted to the last eight intron nucleotides. Completion of the second step leads to the cross-linking of an unidentified ∼80-kDa protein near the branch site sequence, suggesting a potential role for this protein in a later step in intron metabolism. Taken together, these data provide a detailed portrayal of the dynamic associations of proteins with the branch site-3′ splice site region during spliceosome assembly and catalysis.