Functional dissection of 5' and 3' extragenic control regions of human tRNA(Val) genes reveals two different regulatory effects.
Two natural human tRNA(Val) genes, pHtV1 and pHtV3, differ in their transcription efficiency by an order of magnitude. The extragenic control regions (ECRs) responsible for this effect were compared with respect to the kinetics and thermodynamics of transcription complex formation. The 5' ECR of pHtV1 acts by increasing both the rate of stable complex formation and the equilibrium constant of association between tDNA and at least one transcription factor present in the stable complex. The stability of the preinitiation complexes is not affected by ECRs. For the formation of a stable preinitiation complex, we suggest a two-step mechanism, comprising (i) the ECR-controlled association of at least one transcription factor (TFIIIC) with the tDNA, and (ii) an ECR-independent conformational change of this tDNA-protein complex. The function of 3' ECRs could be discriminated from the 5' ECR-mediated effects by transcriptional analysis of two chimeric constructs derived from pHtV1 and pHtV3. Surprisingly, the pHtV1 3' ECR causes an eight-fold increase of transcription efficiency, although it has only minor influence on stable preinitiation complex formation. Instead, this ECR stimulates transcription by promoting the transition of the preinitiation complex into an activity synthesizing transcription complex. This novel function of a 3' ECR contributes an additional regulatory level for tRNA gene expression.
Arnold
G J
GJ
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Wüzburg, FRG.
Schmutzler
C
C
Gross
H J
HJ
D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012366:RNA, Transfer, Val / Q000235:genetics*; D013696:Temperature; D014158:Transcription, Genetic
D012345:RNA, Transfer, Amino Acid-Specific; D012366:RNA, Transfer, Val