Download Structural Biology Tools for Probing Protein/Nucleotide

Structural Biology Tools for Probing Protein/Nucleotide Interactions
Jason P. Rife and Glen E. Kellogg
Dept. of Medicinal Chemistry
We are part of an institute whose primary mission is to understand the principles of biological action by
studying the three-dimensional structures of large biological molecules (RNA, protein, and DNA) and in
complex with ligands (drugs, for example). From these studies we gain further insight into biology, evolution,
and drug action. Others use structural information to advance the fields of biotechnology and bioengineering.
The first 3-D structures determined were of the related proteins hemoglobin and myoglobin. That was in 1957.
Since that time about 20,000 other protein structures have been determined. It is expected that another thousand
structures will be reported just this year. In 1973 the first structure of an RNA molecule, tRNAphe, was
reported. Since that time another dozen or RNA structures have been reported, but usually only in complex with
one or more proteins - tRNA and components of two ribozymes (RNA enzymes) are the only exceptions.
Currently, about a dozen RNA-protein structures have been determined by x-ray crystallography. The largest is
of a 50S ribosomal subunit, which contains two RNA molecules (about 3000 nucleotides) and 30 proteins.
Countless RNA-protein complexes take place in all organisms and form the fundamental basis to many
viruses. Ribosomes are found in all life, often in multiple forms - two exist in humans, one type in the
cytoplasm and one in the mitochondria. These 'amino-acid polymerases' are perhaps the most complicated of all
RNA-protein complexes. All human mRNA must undergo extensive post-transcriptional processing, including
the excision of introns. Introns are excised by a large still poorly understood complex termed the spliceosome.
The spliceosome is composed of numerous RNA (not including the mRNA) and proteins. HIV viral particles
are composed of a collection of proteins and two copies of an RNA molecule that encodes for the manufacture
of new HIV proteins. The assembly and expression of HIV RNA is absolutely dependent on RNA-proteins
interactions, any one of which could be targets of novel HIV therapeutics.
Despite the large collection of known
RNA-protein complexes, the fundamental forces
that govern RNA-protein assembly are poorly
described. A model system to study such forces
is the U1A/U1A binding protein complex that is
part of the larger spliceosome complex. This
particular interaction has several features that
make it an attractive model complex: essential
components are relatively small - a single U1A
binding protein and a small portion of the larger
U1 protein, a relatively high-resolution x-ray
structure, and an interface that is characterized
by a variety of forces - polar and non-polar. In
collaboration with Anne Baranger from
Wesleyan University we hope to fully (or nearly
so) deconvolute those contributing forces from
the multi-pronged approach of mutational
analysis, x-ray crystallography, and molecular
modeling. It is expected that knowledge of such
information will help explain the nature of this
particular interaction as well has help describe
the energetics of other RNA-protein interactions.
The 50S Ribosome.