Localization of transcriptional regulatory elements and nuclear factor binding sites in mouse ribosomal protein gene rpL32.

Abstract

The DNA sequences required for expression of the ribosomal protein gene rpL32 were identified by transient-expression assays of chimeric rpL32-chloramphenicol acetyltransferase genes. These studies showed that maximal rpL32 expression requires sequences in a 150-to 200-base-pair region spanning the transcriptional start site. Three discrete regions of importance were identified: one between positions -79 and -69 and two others located downstream of the transcriptional start site. Progressive 5' or 3' deletions caused stepwise decreases in expression, which suggested a complex interplay of redundant or compensatory elements. Gel mobility shift assays were used to idenfify trans-acting nuclear factors which bind to segments of the rpL32 promoter that are known to be important for transcription. Evidence for several distinct nuclear factors is presented. The binding sites for these factors were localized to the following regions: -79 to -69, -36 to -19, -19 to +11, +11 to +46 in exon I, and within the first 31 base pairs of intron 1. One of these factors may bind to multiple sites within the promoter region. Interestingly, the factor that binds to a sequence motif in the first exon also binds to similar motifs in a comparable region of the c-myc gene.The ribosomal proteins are structurally diverse and evolutionarily unrelated to each other, yet the genes that encode them are expressed with very similar efficiencies, as indicated by the relatively uniform abundance of various ribosomal protein mRNAs and by the similar RNA polymerase loading of different ribosomal protein genes (11, 12; D. E. Kelley and R. P. Perry, unpublished observations). What is the basis for this similarity? Presumably, the transcriptional efficiency of a ribosomal protein gene should ultimately be determined by the characteristics of its particular set of regulatory elements and by the cellular content of transacting factors that interact with these elements. To improve our understanding of these elements and factors, we have sought to define the DNA sequences that constitute the complete promoter of the ribosomal protein gene rpL32 and to identify the nuclear factors that bind to the functionally important sequences. Using a series of constructs in which selected portions of the rpL32 promoter region were linked to a chloramphenicol acetyltransferase (CAT) reporter gene, we observed that maximal expression requires sequences in a 150-to 200-base-pair (bp) region spanning the transcriptional start site, including sequences within exon I and intron 1. Moreover, we show that there are several discrete nuclear factor binding sites within this region, at least one of which recognizes a factor that is also bound by the myc gene promoter. These findings, together with previous and other parallel studies of rpL32 expression (2, 5; R. Moura-Neto, K. P. Dudov, and R. P. Perry, Proc. Natl. Acad. Sci. USA, in press), have delineated the complex set of sequences that determine the efficiency of rpL32 transcription. The unusual organization of this promote...

Equipotent mouse ribosomal protein promoters have a similar architecture that includes internal sequence elements.

“…Both sites embrace the sequence 5'-'^GCGCSGCCATCT-3'. Interestingly, the sequence 5'-GGCGGCCATC-3' is also present in an adjacent intron segment of rpL32, which effectively competes with both rpL30 and rpL32 exon I segments for 8-factor binding and which, itself, binds a factor that was previously termed e (Atchison et al 1989). We suspect that 8 and e may be the same factor and, therefore, that rpL32 actually contains two 8-factor sites separated by -32 bp or three helical turns.…”

“…Boxes marked with X indicate segments that exhibit complex patterns in gel mobility-shift analysis. The data for rpL32 and rpS16 are described in Atchison et al (1989) and Hariharan and Perry (1989), respectively. To have a uniform nomenclature for presumably identical factors, the rpL32 factors previously designated as a and 3 are renamed 3 and 7, respectively.…”

“…5D, lane 7) contains the binding site designated as e (Fig. 4), which was localized to the + 53 to 4-77 region of rpL32 (Atchison et al 1989). Binding to the L32-e site is effectively competed out by fragments containing the 8-factor site from either rpL30 or rpL32 but not by rpL30 fragments containing the 7-factor site or the + 50 to + 79 region of intron 1 (data not shown).…”

“…These single-copy genes are located on different chromosomes (Wiedemann et al 1987) and are evolutionarily unrelated, as judged from the complete dissimilarity of their sequences (Dudov and Perry 1984;Wiedemann and Perry 1984;Wagner and Perry 1985). In this paper, we present a detailed analysis of the rpLSO promoter and compare its properties with those of rpL32 (Dudov and Perry 1986;Atchison et al 1989;Chung and Perry 1989;Moura-Neto et al 1989) and rpS16 (Hariharan and Perry 1989).…”

CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

“…Alternatively, these discrete protein binding sites may be integral parts of a promoter that extends into the gene, such that their orientations and positions relative to the 5Ј flank must be maintained. Intragenic regulatory sequences that operate in this manner have been identified within the genes for c-myc (49) and ribosomal protein L32 (1,13). A final possibility is suggested by the fact that part of the CpG island associated with the rabbit ␣-globin gene coincides with these positive-acting internal sequences.…”