Biomedical subjects

Anthony L Guerrerio

Publications and source records attributed to Anthony L Guerrerio.

4 recordsLinked to original sources

PubMed2004

Metal ion affinities of the zinc finger domains of the metal responsive element-binding transcription factor-1 (MTF1).

Metal response element (MRE) binding transcription factor-1 (MTF1) is a six Cys(2)His(2) zinc finger-containing transcription factor required for basal and zinc-induced transcription of metallothionein genes. The cobalt(II) and zinc(II) affinities of a protein fragment comprising the six zinc finger domains have been examined to reveal apparent dissociation constants (for the six domains collectively) of 0.5 +/- 0.2 microM for cobalt(II) and 31 +/- 14 pM for zinc(II). Two approaches have been used to determine the metal ion affinities of the individual domains. First, the six domains have been examined as single domain peptides revealing dissociation constants ranging from 0.3 to 1.7 microM for cobalt(II). The domains fall into two sets with peptides corresponding to domains 2, 3, and 4 showing relatively high affinity (K(d)(Co(II)) 0.3-0.5 microM) and peptides corresponding to domains 1, 5, and 6 showing lower affinity (K(d)(Co(II)) 1.6-1.7 microM). Second, we examined the affinity of each domain in the context of the six zinc finger domain protein by individually mutating one metal-binding His residue to Cys to allow independent monitoring of the cobalt(II) occupancy of each site. The affinity of each domain was higher in this context than as a single domain peptide with affinities (corrected for the effect of the mutation) ranging from 0.02 to 0.5 microM. The increase in affinity for the individual domains ranged from factors of 1.1 to 20. The order of affinities (from higher to lowest) was observed to be 4 > 2 approximately 5 > 6 approximately 3 approximately 1. These results reveal that none of the Cys(2)His(2) zinc finger domains of MTF1 have dramatically low metal ion affinities, certainly none low enough to respond to changes in free zinc ion concentrations in the micromolar range. Nonetheless, the metal ion affinities of some domains do differ by a factor of 25 with domains at both the amino- and carboxyl-termini showing lower intrinsic affinities for metal ions than the central domains.

Amino Acid Sequence↗

PubMed2003

Selective RNA binding by a single CCCH zinc-binding domain from Nup475 (Tristetraprolin).

Regulation of gene expression takes place at several different levels and involves specific domains involved in specific protein-nucleic acid interactions. The protein Nup475 (also known as Tristetraprolin and TS11) binds to AU-rich sequence elements in certain mRNA molecules and favors the degradation of these mRNAs. The nucleic acid binding domain of Nup475 consists of two CCCH zinc-binding domains. A 36-amino acid peptide corresponding to the first of these CCCH domains has been synthesized and characterized. This peptide binds metal ions such as zinc(II) and cobalt(II) with affinities comparable to those of other authenticated zinc-binding domains. The zinc(II) complex of this peptide binds the RNA oligonucleotide UUUAUUU labeled with fluorescein on the 3'-end with an affinity of approximately 5 microM and discriminates against other sequences lacking the central A or the flanking U residues. These results demonstrate for the first time that a single CCCH domain is capable of binding single-stranded RNA with considerable affinity and selectivity. The combination of this well-behaved domain and the fluorescence-based binding assay sets the stage for more detailed structure-activity studies.

Anisotropy↗

PubMed2003

Correlating structure and affinity for PEX5:PTS1 complexes.

Many proteins that are destined to reside within the lumen of the peroxisome contain the peroxisomal targeting signal-1 (PTS1), a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO(-). The PTS1 is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the energetics of PTS1 binding specificity and to correlate these with features from the recently determined structure of a PEX5:PTS1 complex, we used a fluorescence-based binding assay that enables the quantitation of the dissociation constants for PTS1-containing peptide complexes with the TPR region of human PEX5. Through application of this assay to a collection of pentapeptides containing different C-terminal tripeptide sequences, including both natural and unnatural amino acids, the thermodynamic effects of sequence variation were examined. PTS1 variants that correspond to known functional targeting signals bind to the PEX5 fragment with a change in the standard binding free energy within 1.8 kcal mol(-1) of that corresponding to the peptide ending with -Ser-Lys-Leu-COO(-). The results suggest that a binding energy threshold may determine the functionality of PTS1 sequences.

Amino Acid Substitution↗

PubMed2002

An mRNA surveillance mechanism that eliminates transcripts lacking termination codons.

Translation is an important mechanism to monitor the quality of messenger RNAs (mRNAs), as exemplified by the translation-dependent recognition and degradation of transcripts harboring premature termination codons (PTCs) by the nonsense-mediated mRNA decay (NMD) pathway. We demonstrate in yeast that mRNAs lacking all termination codons are as labile as nonsense transcripts. Decay of "nonstop" transcripts in yeast requires translation but is mechanistically distinguished from NMD and the major mRNA turnover pathway that requires deadenylation, decapping, and 5'-to-3' exonucleolytic decay. These data suggest that nonstop decay is initiated when the ribosome reaches the 3' terminus of the message. We demonstrate multiple physiologic sources of nonstop transcripts and conservation of their accelerated decay in mammalian cells. This process regulates the stability and expression of mRNAs that fail to signal translational termination.

3' Untranslated Regions↗