RF: DNA and RNA Binding Proteins

• helix-turn-helix

• homeodomain/POU domain

• zinc finger

• leucine zipper

• helix-loop-helix

• TATA-box binding protein

Helix-turn-helix

• helix 1: sequence-specific recognition (major groove)

  • palindromic sequence

• helix 2: stabilization, oriented in a 90° angle to helix 1

• additionally, there may be unspecific interactions in the minor groove, e.g. between phosphate groups of the DNA and N-H groups of the protein, and distortion of the DNA

• usually homodimers

Examples: bacteriophage regulatory proteins (repressor for lysogenic cycle, cro for lytic cycle), trp repressor (repressor is inactive unless Trp binds to it – Trp de-novo synthesis gene is suppressed), CAP (catabolite gene activation protein, controls operon for lactose degradation, made of 2 HTH motives, bends the DNA)

• eukaryotic variant of HTH motive: 180 bp coding for 60 aa

• 3 helices:

  • helix 3: sequence-specific recognition

  • additional unspecific interactions with minor groove

• usually heterodimers

• zinc ion coordinated by four residues

• 2 β-strands and one distorted 310-helix, 2 residues of each contribute to the coordination

  • 3-10-helix: right-handed, 3 aa make one turn (one amino acid less than in α-helix)

• helix is responsible for binding, ion only has structural function

• 3 classes:

  • Cys-Cys-His-His (classical)

  • Cys-Cys-Cys-Cys (glucocorticoid receptor)

  • Cys-Cys-His-Cys (HIV p55)

• two helices with hydrophobic residues at one side à zipper-like interactions

• zipper-region and DNA-interaction region

• always homo-or heterodimers

• example: GCN4

• 10-stranded β-sheet with internal symmetry (looks like a dimer)

• bends DNA strand

• RNA processing (modification, splicing)

• translation

• RNA export

• RNA interference

4-thiouridine (s4U) at position 8 of tRNAs (4-thiouridine synthetase)

• stabilization of the RNA fold

• UV sensor (cross-link to other base)

methylation of C38 of tRNA (m5C38) (Dnmt2)

• protection against stress-induced ribonuclease cleavage

• promotion of correct translation (cognate codon recognition)

queunine at position 34 (tRNA guanine transferase)

• only in tRNAs of Asp, Asn, His, Tyr

• necessary for effective action of Dnmt2

• special because the base is completely exchanged, not only modified

• consists of 3 domains:

  • pyrophosphatase domain (PPase, adenylates U8 to activate it, requires ATP)

  • N-terminal ferredoxin-like domain (NFLD)

  • THUMP (RNA binding domain)

• forms homodimer: NFLD and THUMP of monomer 1 bind the tRNA’s acceptor stem loop, PPase of monomer 2 modifies U8

• Structure:

  • (αβ)8 barrel with Zn-finger

  • homodimer in bacteria (both are catalytically active), heterodimer in eukaryotes (only one monomer is active)

snoRNPs

• small nucleolar ribonucleoprotein particles

• involved in ribosome assembly

• can have several functions

  • pseudouridylation

  • methylation of rRNA

  • pre-rRNA processing

  • chaperone-function

• The 2’-OH group of a nucleotide in the intron, the branchpoint, nucleophilically attacks the 5’-end of the intron.

• The 3’-OH group of the “free” exon nucleophilically attacks the last nucleotide of the intron (3’-end), leading to the expulsion of the intron.

• For every pre-mRNA, the spliceosome must be assembled.

• Spliceosome assembly:

  • 5’ splicing site (between 5’-exon and intron) is recognized by U1snRNA

  • branching point (within the intron) is recognized by U2 snRNA

  • Structural rearrangements à activation of the spliceosome

• spliceosome contains metal ions

Disease associated with mutations of proteins involved in splicing

• leads to loss of motor neurons due to

  • SMN1 defect (SMN: survival of motor neuron)

  • SMN2 incorrect splicing – only 10-20% functionality

Drug development: antisense oligonucleotide that modulates splicing of SMN2-mRNA

• threonine, aspartate, glutamate, and arginine are involved

  • Thr and Asp coordinate Mg2+ ion

  • Mg2+ ion and Arg interact with β- and γ-phosphate of the ATP

  • glutamate helps positioning a water molecule that nucleophilically attacks the γ-phosphate

• in spliceosomal DEAD/H-box helicases, 1 ATP is necessary to disrupt 1 bp (in dsRNA)

Processive helicases “walk” along a double strand and separate it on the way. Non-processive helicases “sit” on a double strand and only open a window at that specific location.

Universal mechanism: Thr and Gly coordinate γ-phosphate of the GTP that is to be cleaved off and act like a spring:

Small GTPases do not have an arginine finger themselves (displayed in picture on the right) and therefore have only very low enzymatic activity! They can be activated by GAPs (GTPase activating proteins) which provide the missing Arg finger.

• IF2

• EF-G

• EF-Tu

  • function: delivering correct tRNA to the ribosome

  • has two RNA-binding domains and a GTPase domain

  • no Arg finger necessary! Instead, an rRNA pushes a His in the required position

  • GTP only gets hydrolyzed when tRNA anticodon and mRNA codon fit together

  • EF-Tu dissociates when GDP is bound

• TF

• eIF5B

  • required for subunit joining

  • When the big subunit joins, GTP is hydrolyzed

  • upon GTP hydrolysis, conformational change occurs, leading to a lower affinity to the ribosome

Translational GTPases usually contain a M+ cation as a structural cofactor which might be the reason why they do not need the Arg finger.