BMB 170 Lecture 12 Nucleic Acids, Nov 6Th (Make-Up)

BMB 170 Lecture 12 Nucleic Acids, Nov 6th (make-up)

hp://www.bangroup.ethz.ch/research/nomenclature-of-ribosomal-proteins.html Ban N, Beckmann R, Cate JH, Dinman JD, Dragon F, Ellis SR, Lafontaine DL, Lindahl L, Liljas A, Lipton JM, McAlear MA, Moore PB, Noller HF, Ortega J, Panse VG, Ramakrishnan V, Spahn CM, Steitz TA, Tchorzewski M, Tollervey D, Warren AJ, Williamson JR, Wilson D, Yonath A, Yusupov M. ‘A new system for naming ribosomal proteins.’ Curr Opin Struct Biol. 2014;24:165-9. doi: 10.1016/j.sbi.2014.01.002. Jun NFAT/Fos-Jun/DNA Fos

NFAT

• AP-1 heterodimer - Fos and Jun (both bZIP proteins) • NFAT (nuclear factor of activated T cells) has RHR DNA-binding region • Structure bZIP parts of AP-1 and RHR of NFAT bound to DNA fragment from Interleukin-2 promoter • NFAT binds as a monomer, other RHR members (e.g., NFκB) are dimers Harrison lab (1a02) Chen et al (1998) Nature 392: 42-8. MEF2/Cabin1/DNA has MHC-like interaction

§ MEF2 (myocyte enhancer-2) family of transcription factors involved in development and function of T cells, neuronal cells, muscle cells

§ Human MEF2B contains sequential MADS box and MEF2-speciﬁc domain (MEF2S)

§ Co-repressors of MEF2 include Cabin1/Cain and class II histone deacetylases

Han et al Nature (2003) 422: 730-4 Crystal structure of MEF2/Cabin1/DNA complex (1n6j)

Cabin1

MEF2

• Crystal structure of MEF2B and MEF2 binding motif of Cabin1 • MADS box – helix followed by 2 strands – SRF and MCM1 • MEF2S domain – helix-sheet-helix – not folded without partner • Binding resembles MHC peptide pocket

Han et al Nature (2003) 422:730-4 Transcriptional regulation of eukaryotic genes

• Basal promoter elements – e.g., TATA box – 25 bp upstream of transcription start site • Promoter proximal element – 100-200 bp long (close to site initiation) – Contains sequences recognized by different transcription factors • Enhancer elements – Can be a few thousand to 20,000 bp upstream or downstream from the initiator site Transcriptional activation involves interactions over long stretches of DNA

• TATA box binding protein (TBP) – part of TFIID – binds to RNA pol and other proteins to form pre-initiation complex • Pre-initiation complex interacts with different speciﬁc transcription factors bound to promoter proximal elements and enhancer elements • Each gene in every cell has same DNA control sequences, but not every cell has complete set of DNA binding proteins to turn on every gene Human pre-iniaon complex

He, Fang, Taatjes, Nogales. “Structural visualization of key steps in human transcription initiation.” Nature (2013) 495:481-6 Human pre-iniaon complex

He, Fang, Taatjes, Nogales. “Structural visualization of key steps in human transcription initiation.” Nature (2013) 495:481-6 Structures of transcripon pre-iniaon complex with TFIIH and Mediator

Schilbach..Cramer Nature (2017) doi:10.1038/nature24282 TATA-box binding protein (TBP)

• Initiation of gene transcription by RNA pol requires action of general factors that form a specific multiprotein complex near the transcription start site • TFIID binds to TATA box, then 5 other general factors bind. TFIID consists of universal TBP and other associated factors that bring in species and cell- specific variations Structure of TBP alone solved first (1tbp)

Curved 10-stranded antiparallel β-sheet with four helices on top

Looks like saddle

Originally assumed that concave surface of saddle would sit astride DNA double helix

Model of DNA bound to TBP (not crystal structure) Burley lab, Nikolov et al Nature (1992) 360: 40-46 Structure of TBP bound to DNA

• Novel dramatic conformational • TATA element unwinds by ~110˚ change – changes in bend and twist • Binds across minor groove compensate • TBP bends DNA ~80˚ – no net change in linking number

Sigler lab, Kim, Y et al Nature (1993) 365: 512-20 (1ytb); Burley lab, Kim, J.L. et al, ibid. 520-7 (1tgh) Structure of TBP bound to DNA

Oligo in crystal structure

Modeled DNA

Minor groove of TATA box is widened and ﬂaened -- interacts with enre concave underside of TBP saddle

Sigler lab, Kim, Y et al Nature (1993) 365: 512-20 (1ytb); Burley lab, Kim, J.L. et al, ibid. 520-7 (1tgh) Transcripon factor IIA (TFIIA) plus TBP bound to TATA element

cyTBP

cyTOA1C

cyTOA2

cyTOA1N • Structure of core yeast TFIIA and TBP • TFIIA (TOA1/TOA2) composed of β-barrel and four helix bundle. • β-barrel of TFIIA – extends TBP β-sheet – crosses DNA major groove upstream of TATA box • Bundle interacts with other transcription factors Richmond lab, Tan et al Nature (1996) 381: 127-34 (1y) Cooperative binding between separated sites on a promoter

• c-Myb (proto-oncogene product) cooperates with C/EBPβ (CAAT- enhancer binding protein β) to regulate transcription of myeloid- speciﬁc genes • c-Myb and C/EBPβ promoters are far apart – interact and cooperate via DNA looping • Crystal structure of c-Myb and C/ EBPβ and DNA fragment

Tahirov et al Cell (2002) 108:57-70 c-Myb/ C/EBPβ / DNA C/EBPβ structure (1h88)

• C-terminal fragment of C/ EBPβ is a bZIP protein dimer • DNA binding domain of c- R3 Myb contains three homeodomains (R1, R2, R3) c-Myb • Structure contains DNA with both binding sites

Tahirov et al (2002) Cell 108:57-70 (1h88) R1 C/EBPβ C/EBPβ and c-Myb interaction • Coiled-coil region of C/EBPβ binds α1 and α2 helices of c-Myb R2 on R1 different DNA fragment - forms four- c-Myb helix bundle • Exposed hydrophobic patch on R2 of c-Myb involved in interaction with C/ EBPβ R2 R3 • Corresponding region of R2 of v-Myb has three substitutions, so v-Myb doesn’t cooperate with C/EBPβ to regulate transcription

Tahirov et al (2002) Cell 108:57-70 (1h88) Atomic Force Microscopy (AFM) shows interaction between c-Myb and C/EBPβ bound at a distance from one another on the mim-1 promoter. (C/EBPβ site at -65; c-Myb site at -147)

C-Myb and C/EBPβ bind to co-activator CBP through their transcriptional activation domains

Multiple enhancer binding proteins and co-activators then interact to form “enhanceosome”. Capacity of transcription factors to interact at a distance facilitates formation of enhanceosome. Tahirov et al (2002) Cell 108:57-70 ZFNs: Zinc ﬁnger chimeric endonucleases

• Link Zn fingers to the nonspecific cleavage endonuclease domain of FokI restriction enzyme (a dimer) • Use four fingers per FokI monomer – 8 Finger recognition sequence – effectively have a 24-bp recognition site – long enough to specify unique site in mammalian genomes • Zn fingers recognition identified by phage display – GNN triplets (Kim lab LBNL - Jamieson et al PNAS (1996) 93:12834-9) – CNN triplets – most of rest are in proprietary database of Sangamo BioSciences (Patent app# EP20010987037) • Use for gene therapy – Urnov et al Nature (2005) 435: 646-51 (N&V, pp 577-9) – Kandavelou et al Nature Biotech (2005) 23: 686-7 – Currently sold by Sigma-Aldrich High (2005) Nature 435:577-9 Sequence recognition

• Dervan Lab • Polyamides – Small molecules that bind speciﬁcally to DNA with high afﬁnity – Bind in minor groove

Kielkopf et al NSB (1998) 5:104 (365d) Can design inhibitors

Dervan & Edelson COSB (2003) 13:284 Genomes get big

Organism Genome Size Genes HIV 9,700 9 M. genitalium 580,000 473 E. coli 4,700,000 4100 H. sapiens 3,000,000,000 ~20,000 Amphibians >80,000,000,000 Plants >900,000,000,000 Nucleosome • First proposed by Roger Kornberg (1974) • Provides the ﬁrst level of compaction • Regulates access to genes by RNA polymerase • 4 “core” histones – H2A, H2B, H3 and H4 – Assemble into octamer • 145 BP of DNA wrapped twice (1.65 left handed superhelical turn) – 200 BP protected H3-H4 tetramer binds two H2A-H2B dimers to form the histone octamer

Monomers

Dimers

Octamer

Tetramer Nucleosome core particle (1aoi)

• Nucleosome has pseudosymmetry • 147 bp of DNA wrapped almost twice core histones (1.65 left handed superhelical turn) • DNA bent at major groove face • Interacts ~every 10BP • Histone tails extend out to regulate DNA binding

Richmond Lab: Luger et al (1997) Nature38:251-60 Nucleosome packing ain’t so simple

“Cryo-ET reveals nucleosome reorganizaon in condensed mitoc chromosomes in vivo” Cai, Chen, Tan, Shi & Gan bioRxiv (2017) RNA binding proteins • RNA – Steric clashes force A-form to dominate – Can form complicated tertiary structure • Large complexes – Spliceosome – Ribosome • Families – Arg-rich motif (Tat & Rev) – All helical (Rop & L11) – αβ protein domain • RNA recognition motif RRM (U1A, PABP, HuD) - over 6000 • double-stranded RNA-binding motif dsRBM (Rnase III) • K-homology domain KH (Nova) • Piwi Argonaut Zwille domain (PAZ) – Zn-ﬁnger (TFIIIA, HIV-1 NC) – OB fold (S12, S17)

Reviewed in Chen & Varani FEBS Journal (2005) 272:2088-97 Spliceosomal U1A protein -RRM

• Component of U1 small nuclear RNP complex • Pre-mRNA splicing • U1A ﬁrst RNA-binding motif structure • RNA recognition motif – RRM – RNA binding platform – Contains RNP motif – βαββαβ fold

Nagai lab, Nagai et al Nature (1990) 348:515-20 (1oia) Oubridge et al Nature (1994) 372:432-8 (1urn) αβ RNA binding domain

U1A Nova KH Rnase III - dsRBM

Chen & Varani FEBS Journal (2005) 272:2088-97 α/β fold interactions in ribosome S6

S11 S3

Brodersen et al JMB (2002)316:725-68 Glu synthetase

Recognition elements

Fig. 8.4 & 8.6 Class I Class II Class I: Glu synthetase/ tRNA complex

Fig. 8.7 Class II: Asp synthetase/tRNA complex

Fig. 8.8 Ribosome structures

Together comprise over 50 proteins and over ~5000bp of RNA • 70S subunit – Catalyzes protein synthesis in all organisms Ribosomes – 2.5 Megadaltons in bacteria • Small subunit – 30S in bacteria (40S in eukaryotes) – 16S rRNA (~1500 nucleotides) – ~20 proteins – First complete atomic structure by Ramakrishnan lab (2000) from Thermus thermophilus • Large subunit – 50S in bacteria (60S in eukaryotes) – 23S rRNA (~2900 nucleotides) & 5S rRNA (~120 nucleotides) – ~30 proteins – First complete atomic structure by Steitz & Moore (2000) from the archaea Haloarcula marismortui Early EM model from Lake lab (UCLA)

Component Structures S19 S7*

S4*

S17*

S5*

S16* S15* S8* Crystallizaon of 30S from Thermus thermophilus

Trakhanov et FEBS Lett.220, 319 (1987) MPD - al.

Glotz et al. Biochem. Int. 15, 953 Ethylbutanol - (1987) /ethanol Yonath et al. J. Mol. Biol. 203, 831 MPD 9.9 Å (1988) Yusupov et al. FEBS Lett. 238, 113 (1988) MPD 12 Å Head

The 5.5Å Map Plaorm

Shoulder

Body The 5.5 Å structure of the 30S

•First architectural model •7 known proteins placed •Central domain of RNA •H44 & decoding site •S20 Prescreening to ensure crystal uniformity

27 ribosome preps (2 weeks each) 81 trays set up ~1030 crystals frozen ~400 crystals screened 23 complete data sets (~10 crystals each) Our 20Model Proteins 1 Large RNA

Reﬁnement Percent excluded for cross-validaon: 5% R/Rfree: 0.208/0.254 Deviaon from ideality: Bond lengths: 0.006 Å Angles: 1.24 degrees Front Back Subunit Interface Solvent Accessible Head

Beak Platform

Shoulder

Body

Spur The 16S RNA 3’ Major Head

Central Beak Plaorm

Shoulder

Body 5’ 3’ Minor

Spur 23S RNA

Lot’s of A-minor type of interactions Ribosomal proteins (1jfe & 1ffk)

Ramakrishnan & Moore Curr Opin Str Biology (2001) 11:144-54 S12 connects front to back Tth 70S Structure

• First high-resoluon structure of the 70S • Contains mRNA, AP &E site tRNAs • 1j00 & 1j01 (2 molecules in the asymmetric unit) Ramakrishnan Lab: Selmer et al Science (2006) 313:1935 Ribosome Architecture

Review: Schmeing & Ramakrishnan (2009) Nature