Unveiling DNA Binding Proteins: Mechanisms and Structures in Transcription Initiation

1. Chapter 11:Assembly of the Transcription Initiation Complex Transcription is more complex than once assumed.

4. 11.1 DNA-binding proteins and their attachment sites Direct readout

5. 11.1.1 The special features of DNA-binding proteins • X-ray crystallography • nuclear magnetic resonance (NMR) spectroscopy • A limited number of motifs

7. The helix-turn-helix motif is present in prokaryotic and eukaryotic proteins • Beta-turn, made up of four amino acids. • The HTH structure is usually 20 or so amino acids in length, so just a small part of the protein as a whole. • Many proteins eukaryotic and prokaryotic, including lactose repressor and homeodomain proteins.

8. Zinc fingers are common in eukaryotic proteins • Rare in prokaryotic proteins but very common in eukaryotic proteins. • Multiple copies of the finger are sometimes found on a single protein.

9. 11.1.2 The interaction between DNA and its binding proteins • Direct readout of the B-form DNA predominantly involves contacts in the major groove.

10. Something New Protein–DNA recognition • The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanism: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. • Rohs et al. report that the shape of the minor groove of DNA can direct the binding of proteins to specific sites. 1. Rohs R, West SM, Sosinsky A, Liu P, Mann RS, Honig B: The role of DNA shape in protein-DNA recognition. Nature 2009, 461:1248-1253. 2. Tullius T: Structural biology: DNA binding shapes up. Nature 2009, 461:1225-1226.

11. Something New Protein–DNA recognition • a, Negatively charged phosphate groups (magenta) are arrayed along the outer edge of the DNA major and minor grooves that spiral around the axis of the double helix. The width of the minor groove varies depending on the sequence of nucleotides. This variation leads to differences in the distance between phosphates across the groove, which in turn lead to variation in the negative electrostatic potential along the minor groove. b, A representation of a DNA-binding protein (green) that has a positively charged side chain on its surface, for example arginine (purple), is shown. The protein may recognize a binding site on DNA by its electrostatic potential. The protein is about to bind to the segment of the DNA minor groove that has the optimum groove width and electrostatic potential for binding (red).

12. The nucleotide sequence has a number of indirect effects on helix structure • It is possible for the A-, B- and Z-DNA configuration, and intermediates between them, to coexist within a single DNA molecule, different parts of the molecule having different structures. • These conformational variations are sequence dependent, being largely the result of the base-stacking interactions that occur between adjacent base pairs. • At present this is just a theoretical possibility. • DNA bending at specific sequence regions.

13. Contacts between DNA and proteins • Most proteins that recognize specific nucleotide sequences are also able to bind nonspecifically to other parts of a DNA molecule. • Specific form of binding is more favorable in thermodynamic terms than nonspecific ones. • Many DNA-binding proteins are dimers.

14. 11.2 DNA-protein interactions during transcription initiation Archaea Bacteria Chloroplasts Mitochondria

15. 11.2.2 Recognition sequences for transcription initiation

16. Bacterial RNA polymerases bind to promoter sequences

17. Eukaryotic promoters are more complex

18. 11.2.3 Assembly of the transcription initiation complex

19. Transcription initiation in E. coli submit2

20. Transcription initiation with RNA polymerase II • RNA polymerase II does not directly attached the DNA

22. 11.3 Regulation of transcription initiation

23. 11.3.1 Strategies for controlling transcription initiation in bacteria • Constitutive control, by promoter sequences • Regulatory control, by proteins

28. Riboswitches • The switching on and switching off of genes in response to an organism's needs is one of the most basic of biological control mechanisms. When François Jacob and Jacques Monod were mapping out the genetic regulatory circuit that controls metabolism of a simple sugar (lactose) in a bacterium, they thought for a while that the gene repressor might be RNA. Soon afterwards, however, the lac repressor was isolated – it turned out to be a protein, as were the next hundred or so repressors that were identified. So the idea of an RNA gene-repressor lay dormant for decades.

29. Riboswitches • Very recently, however, this issue has been revisited. In another branch of the bacterial kingdom, RNA elements built into messenger RNAs can directly sense the concentration of small metabolites and turn gene expression on or off in response. These riboswitches fold into intricate structures that can distinguish one metabolite from another. Three distinct tricks for switching gene expression have been revealed: the RNA element can cause premature termination of transcription of the mRNA, it can block ribosomes from translating the mRNA, or it can even cleave the mRNA and thereby promote its destruction. This last activity involves an RNA unit directly binding a small-molecule metabolite, which switches the RNA into a conformation that activates its intrinsic self-cleavage activity. This "ribozyme riboswitch" represents a new type of biological activity for a catalytic RNA.

30. 11.3.2 Control of transcription initiation in eukaryotes • The basal rate of transcription initiation in eukaryotes is very low. • Activators play a much prominent role than repressor proteins. • Eukaryotic promoters contain regulatory modules • Alternative promoter

32. Controlling the activities of activators and repressors • control its synthesis: activators and repressors responsible for maintaining stable patterns of genome expression. • chemical modification, for example by phosphorylation, or by inducing a change in its conformation: • The later changes are much more rapid than de novo synthesis, and enable the cell to respond to extracellular signaling compounds that induce transient changes in genome expression

33. Further readings • Brantl, S 2004. Bacterial gene regulation: from transcription attenuation to riboswitches and ribozymes. Trends Microbiol.12: 473-475. • Mandal, M and Breaker, RR 2004. Gene regulation by riboswitches. Nat. Rev. Mol. Cell Biol.5: 451-463. • Soukup, JK and Soukup, GA 2004. Riboswitches exert genetic control through metabolite-induced conformational change. Curr. Opin. Struct. Biol.14: 344-349. • Tucker, BJ and Breaker, RR 2005. Riboswitches as versatile gene control elements. Curr. Opin. Struct. Biol.15: 342-348. • Vitreschak, AG, Rodionov, DA, Mironov, AA, and Gelfand, MS 2004. Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet.20: 44-50. • Winkler, WC 2005. Riboswitches and the role of noncoding RNAs in bacterial metabolic control. Curr. Opin. Chem. Biol.9: 594-602. • Winkler, WC and Breaker, RR 2005. Regulation of bacterial gene expression by riboswitches. Annu. Rev. Microbiol.59: 487-517. • Razin SV et al. 2011. Transcription factories in the context of the nuclear and genome organization. Nucleic Acids Res 39: 9085-9092.