Protein Nucleic Acid Interaction

Pune University23rd April 2014

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Protein-DNA Interactions : Importance

• Gene expression– Transcription initiation (TATA binding protein)– RNA synthesis (RNA polymerase)– Transcription regulation (MAX protein)

• DNA repair (DNA glycosylase : oxidative DNA damage)

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Protein-DNA Interactions : Importance• DNA packaging (Histone H2A.e)• DNA replication (Polymerases, Ligases, single stranded

binding proteins)

Types of Protein-Nucleic acid interaction

Protein – DNA interactionssDNA

dsDNA

Protein – neucliotide interaction

Protein – RNA interactionmRNA

siRNA

P- Loop (GXXX)

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DNA

• DNA has structural flexibility• Structure described by Watson

& Crick : B-form

B A Z

Feature B A Z

Type of helix RH RH LH

Diameter 1.9 2.3 1.8nm

Rise per bp 0.34 0.29 0.37nm

# bp per turn 10.5 11 12

Major groove

Wide,deep

Narrow,deep

Minor groove

Narrow, shallow

Wide, shallow

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Structural Data• NDB : assemble and distribute

structural information about nucleic acids

• 2490 structures (25/08/04)

Protein-DNA Complex

Number

Double Helix 593Single Strand 57

http://ndbserver.rutgers.eduBerman et al., 1992. Biophys J 63 p751

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Protein-DNA Interactions : Structure

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Protein-DNA Interactions : characteristics

• Major and minor groove binding• DNA-binding motifs• Positively charged surface areas • Size ASA : 618Å2 - 2833Å2 • Conformational changes

– DNA bending– domain movements, quaternary changes

• Nadassy et al., 1999 Biochemistry 38 p1999• Jones et al., 1999 J.Mol.Biol. 287 p877

Binding of Proteins to DNA Often Involves Hydrogen Bonding

• Gln/Asn can form specific H-bond with Adenine’s N-6 and H-7 H’s

• Arg can form specific H-bonds with Cytosine-Guanine base pair

• Major groove is right size for -helix and has exposed H-bonding groups

DNA-binding domains• Proteins generally recognise aspects of nucleic acid

sequence, or variations in structure and/or flexibility• High-resolution structures of many protein-DNA

complexes have now been solved• Similar structural domains occur in different proteins:

Helix-turn-helixZinc-fingerZinc-binding domain Basic region-leucine zipper (bZIP)β-sheet recognition

The Helix-turn-helix Motif is Common in DNA-binding Proteins

• Each “helix-turn-helix” covers ~ 20 aaOne -helix for DNA

recognition, then -turn, then another -helix

Sequence-specific binding due to contacts between the recognition helix and the major groove

• Four DNA-binding helix-turn-helix motifs in the Lac repressor

Helix-turn-helix· Helix-turn-helix is most common observed DNA-

binding unit in prokaryotes

Berg, Tymoczko & Stryer, “Biochemistry”, 5th edn, 2002, p. 874

· Note that 34 Å corresponds to 1 turn of DNA

Zinc-finger· One of best-studied examples of DNA binding domain,

but also binds RNA· Each covers ~30 aa· Binding is relatively weak, so typically there are a series

of zinc fingers· “Finger” portion is a peptide loop cross-linked by Zn2+,

which is usually coordinated by 4 Cys, or 2 Cys + 2 His· One type of consensus sequences is:

“Cys2/His2”: Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His· Zinc is held in tetrahedral structure by conserved Cys

and His

Zn++ Zn++

= Cys

= His

· Regulatory protein Zif268, complexed with DNA

Zinc Finger Motif is Common in Eukaryotic Transcription Factors

β-recognition motif· In some prokaryotic regulatory proteins, this is an

alternative DNA-binding motif

· E. coli methionine repressor binds DNA through insertion of pair of β-strands into major groove

Berg, Tymoczko & Stryer, “Biochemistry”, 5th edn, 2002, p. 874

2% of the human genome contain RRMs

The domain has a typical size of 80-90 amino acids, and consists of two α-helices packed against a four-stranded anti-parallel β–sheet with βαββαβ topology. R or K residue on the signature conserved sequence RNP1 forms a salt bridge with the RNA phosphodiester backbone, two often aromatic residues on RNP1 and RNP2(the second conserved sequence that defines the motif) form stacking interactions with the nucleobases.

Protein RNA interaction

Protein RNA interaction, Pumilio (PUF) repeats

The Code:The combination of cysteine and glutamine binds adenine, asparagine and glutamine bind uracil, serine and glutamate bind guanine, and arginine and serine bind cytosine.

2- 30 RepeatsOne amino acid side chain forms stacking interactions with the aromatic ring of the RNA base, while the other two side chains recognize the Watson–Crick or Hoogsteen edges of the base via base-specific hydrogen bonding and van der Waals contacts.

Protein RNA interaction, Pentatricopeptide repeat (PPR)

Localize primarily to mitochondria and chloroplasts, where they influence various aspects of RNAmetabolism

35 amino acids occurring as tandem arrays of 2–26 repeats per protein, closely related to the tetratricopeptide repeat motifs that mediate protein–protein interactions, PPRs appear to be exclusively involved in RNA–protein interactions.

Protein RNA interaction, CCCH zinc fingers

Role in differentiation, development and other gene regulatory processes ineukaryotes

class II AU-rich elements in the 3 UTR ′of target mRNAs and promoting their deadenylation and degradation via recruitment of the exosome

conserved tandem ZF domain with two CX8CX5CX3H ZF motifs of approximately 35 amino acids separated by fixed linker length

Post-transcriptional regulatory processes, including RNA editing and miRNA bio-genesis and function, as well as RNA export and localization.

Recognize the shape of the A–form helix of dsRNA in a sequence-independent manner.

Protein RNA interaction, dsRNA

Replication Termination