S4-alpha mRNA translation repression complex. I. Thermodynamics of formation

J Mol Biol. 1987 Jul 20;196(2):313-22. doi: 10.1016/0022-2836(87)90692-9.

Abstract

Expression of the four ribosomal proteins from the Escherichia coli alpha operon (S4, S11, S13, and L17) is regulated at the level of translation by the binding of S4 to the alpha mRNA. Using a filter binding assay and alpha mRNA sequences prepared by in-vitro transcription, previous work located the S4 target site within the approximately 100-base leader sequence. We have extended this work to include fragments of the alpha leader with six different 5' end points and four different 3' end points. A core region between bases 23 and 69 (numbering from the first nucleotide of the E. coli transcript) binds S4 with an affinity of approximately 2 microM-1. Regions of weak interactions are located in the 22 nucleotides 5' and the 70 nucleotides 3' to this core; they increase the S4 affinity to approximately 13 microM-1. Studies of S4-alpha mRNA binding under different conditions have revealed the following. (1) Specific and non-specific binding show the same dependence on K+ concentration, with delta log+ K/delta log [K+] approximately 4 in most potassium salts. With KCl and KBr, much weaker salt dependence of specific complex formation is observed suggesting that the protein responds to the correct RNA substrate by binding halide anions. (2) Increasing the MgCl2 concentration between 1 and 4 mM enhances binding by a factor of 4, with no further effects up to 20 mM. About five Mg2+ are taken up by the complex with an average binding constant of approximately 600 M-1 each. Renaturation of the RNA in the presence of MgCl2 is also required to obtain full binding. These effects are seen only with alpha mRNA extending beyond the initiation codon; S4 binding to the alpha leader sequence itself is insensitive to Mg2+. (3) The association kinetics are fast and probably diffusion controlled. (4) Formation of the complex is entirely entropy driven.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Escherichia coli / genetics*
  • Kinetics
  • Magnesium / metabolism
  • Operon
  • Protein Biosynthesis*
  • RNA, Bacterial / metabolism
  • RNA, Messenger / metabolism*
  • Regulatory Sequences, Nucleic Acid*
  • Ribosomal Proteins / metabolism*
  • Thermodynamics*

Substances

  • RNA, Bacterial
  • RNA, Messenger
  • Ribosomal Proteins
  • ribosomal protein S4
  • Magnesium