Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation

GFI is an abundant DNA binding protein in the yeast S. cerevisiae. The protein binds to specific sequences in both ARS elements and the upstream regions of a large number of genes and is likely to play an important role in yeast cell growth. To get insight into the relative strength of the various GFI-DNA binding sites within the yeast genome, we have determined dissociation rates for several GFI-DNA complexes and found them to vary over a 70-fold range. Strong binding sites for GFI are present in the upstream activating sequences of the gene encoding the 40 kDa subunit II of the QH2:cytochrome c reductase, the gene encoding ribosomal protein S33 and in the intron of the actin gene. The binding site in the ARS1-TRP1 region is of intermediate strength. All strong binding sites conform to the sequence 5' RTCRYYYNNNACG-3'. Modification interference experiments and studies with mutant binding sites indicate that critical bases for GFI recognition are within the two elements of the consensus DNA recognition sequence. Proteins with the DNA binding specificities of GFI and GFII can also be detected in the yeast K. lactis, suggesting evolutionary conservation of at least the respective DNA-binding domains in both yeasts.     

Cost minimization of ribosomal frameshifts

Properties of mRNA leading regions that modulate protein synthesis are little known (besides effects of their secondary structure). Here I explore how coding properties of leading regions may account for their disparate efficiencies. Trinucleotides that form off frame stop codons decrease costs of ribosomal slippages during protein synthesis: protein activity (as a proxy of gene expression, and as measured in experiments using artificial variants of 5' leading sequences of beta galactosidase in Escherichia coli) increases proportionally to the number of stop motifs in any frame in the 5' leading region. This suggests that stop codons in the 5' leading region, upstream of the recognized coding sequence, terminate eventual translations that sometimes start before ribosomes reach the mRNA's recognized start codon, increasing efficiency. This hypothesis is confirmed by further analyses: mRNAs with 5' leading regions containing in the same frame a start preceding a stop codon (in any frame) produce less enzymatic activity than those with the stop preceding the start. Hence coding properties, in addition to other properties, such as the secondary structure of the 5' leading region, regulate translation. This experimentally (a) confirms that within coding regions, off frame stops increase protein synthesis efficiency by early stopping frameshifted translation; (b) suggests that this occurs for all frames also in 5' leading regions and that (c) several alternative start codons that function at different probabilities should routinely be considered for all genes in the region of the recognized initiation codon. An unknown number of short peptides might be translated from coding and non-coding regions of RNAs.     

Mechanism of mRNA binding to bovine mitochondrial ribosomes

The binding of mRNA to bovine mitochondrial ribosomes was investigated using triplet codons, homopolymers and heteropolymers of various lengths, and human mitochondrial mRNAs. In the absence of initiation factors and initiator tRNA, mitochondrial ribosomes do not bind triplet codons (AUG and UUU) or homopolymers (oligo(U] shorter than about 10 nucleotides. The RNA binding domain on the 28 S mitoribosomal subunit spans approximately 80 nucleotides of the mRNA, judging from the size of the fragments of poly(U,G) and natural mRNAs protected from RNase T1 digestion by this subunit, but the major binding interaction with the ribosome appears to occur over a 30-nucleotide stretch. Human mitochondrial mRNAs coding for subunits II and III of cytochrome c oxidase and subunit 1 of the NADH-ubiquinone oxidoreductase (complex I) were used in studying in detail the binding of mRNA to the small subunit of bovine mitochondrial ribosomes. We have determined that these mRNAs have considerable secondary structure in their 5'-terminal regions and that the initiation codon of each mRNA is sequestered in a stem structure. Little mRNA was bound to ribosomes in a manner conferring protection of the 5' termini from RNase T1 digestion, under standard conditions supporting the binding of artificial templates, but such binding was greatly stimulated by the addition of a mitochondrial extract. Initiation factors and tRNAs from Escherichia coli were unable to stimulate the 5' terminus protected binding of these mRNA molecules, demonstrating a requirement for homologous factors. Our results strongly suggest that mitochondrial initiation factors are required for the proper recognition and melting of the secondary structure in the 5'-terminal region of mitochondrial mRNAs, as a prerequisite for initiation of protein synthesis in mammalian mitochondria.     

Interaction of the mouse chromosomal protein HMG-I with the 3' ends of genes in vitro

High affinity mouse HMG-I binding sites have been distinguished from other (A+T)-rich sequences using band competition assays. These sites have been found 3' to the coding regions of a variety of genes. For the herpes simplex virus thymidine kinase and minute virus of mice genes, high affinity HMG-1 binding sites were further localized to the functional polyadenylation signal by DNase I footprinting. These results suggest that HMG-I may function at the 3' ends of genes in vivo.     

Inference of positive and negative selection on the 5' regulatory regions of Drosophila genes

Both positive selection and negative selection have been shown to drive the evolution of coding regions. It is of interest to know if the corresponding 5' regions of genes may be subjected to selection of comparable intensities. For such a comparison, we chose the Accessory gland protein (Acp) genes as our test case. About 700 bp and 600 bp for the 5' and coding regions, respectively, of eight previously unstudied genes were sequenced from 21 isogenic lines of D. melanogaster and one line from D. simulans. The ratio of divergence at the amino-acid replacement sites (A) over that at the synonymous sites (S) was twice the ratio for common polymorphism. Interestingly, the 5' region shows the same trend, with the 5'/S divergence ratio being 1.8 times higher than the 5'/S ratio for common polymorphism. There are several possible explanations for the 5'/S ratios, including demography, negative selection, and positive selection. Under normal conditions, positive selection is the most likely explanation. If that is true, about 45 to 50 percent of all fixed differences at both the replacement and 5' sites were adaptive, even though the substitution rate in the former is only half that of the latter (K(A)/K(S) approximately 0.3 vs. K(5')/K(S) approximately 0.6). As previous analyses have indicated, the inclusion of slightly deleterious polymorphism confounds the inference of positive selection. The analysis of published polymorphism data covering 97 verified 5' regions of Drosophila suggests more pronounced selective constraint on the 5' untranslated region and the core promoter (together corresponding to approximately 200 bp in this data set) when compared to the more distal portion of the 5' region of genes.     

Predicted functional RNAs within coding regions constrain evolutionary rates of yeast proteins

Functional RNAs (fRNAs) are being recognized as an important regulatory component in biological processes. Interestingly, recent computational studies suggest that the number and biological significance of functional RNAs within coding regions (coding fRNAs) may have been underestimated. We hypothesized that such coding fRNAs will impose additional constraint on sequence evolution because the DNA primary sequence has to simultaneously code for functional RNA secondary structures on the messenger RNA in addition to the amino acid codons for the protein sequence. To test this prediction, we first utilized computational methods to predict conserved fRNA secondary structures within multiple species alignments of Saccharomyces sensu strico genomes. We predict that as much as 5% of the genes in the yeast genome contain at least one functional RNA secondary structure within their protein-coding region. We then analyzed the impact of coding fRNAs on the evolutionary rate of protein-coding genes because a decrease in evolutionary rate implies constraint due to biological functionality. We found that our predicted coding fRNAs have a significant influence on evolutionary rates (especially at synonymous sites), independent of other functional measures. Thus, coding fRNA may play a role on sequence evolution. Given that coding regions of humans and flies contain many more predicted coding fRNAs than yeast, the impact of coding fRNAs on sequence evolution may be substantial in genomes of higher eukaryotes.     

Inhibition of rabbit beta-globin synthesis by complementary oligonucleotides: identification of mRNA sites sensitive to inhibition

We tested the effects of a series of synthetic oligonucleotides (hybridons) complementary to the 5' noncoding and coding regions of rabbit beta-globin mRNA on endogenous protein synthesis in a rabbit reticulocyte cell-free translation system. With highly purified hybridons inhibition was completely specific for beta-globin. The sites most sensitive to inhibition are the beginning of the 5' noncoding region and a sequence including the initiation codon and several upstream bases. The region between these was relatively insensitive to inhibition. The sites of maximum sensitivity coincide with known protein binding sites, suggesting that hybridons exert their effects in part by blocking the binding of proteins required for translation. Their effectiveness seems related to the ease with which they are displaced by ribosomes.