PDBsum entry 3kit

Ribosome

PDB id: 3kit

Contents

Protein chains

120 a.a. *

271 a.a. *

204 a.a. *

207 a.a. *

181 a.a. *

164 a.a. *

145 a.a. *

130 a.a. *

138 a.a. *

122 a.a. *

146 a.a. *

140 a.a. *

117 a.a. *

98 a.a. *

135 a.a. *

117 a.a. *

101 a.a. *

113 a.a. *

92 a.a. *

100 a.a. *

184 a.a. *

84 a.a. *

93 a.a. *

71 a.a. *

59 a.a. *

57 a.a. *

55 a.a. *

50 a.a. *

47 a.a. *

63 a.a. *

37 a.a. *

DNA/RNA

Metals

_MG ×300

_ZN

* Residue conservation analysis

Obsolete entry

PDB id:

3kit

Name:

Ribosome

Title:

Structure of rele nuclease bound to the 70s ribosome (precleavage state; part 4 of 4)

Structure:

50s ribosomal protein l1. Chain: c. 50s ribosomal protein l2. Chain: d. 50s ribosomal protein l3. Chain: e. 50s ribosomal protein l4. Chain: f. Synonym: l1e.

Source:

Thermus thermophilus hb8. Organism_taxid: 300852. Strain: hb8 / atcc 27634 / dsm 579. Strain: hb8 / atcc 27634 / dsm 579

Resolution:

3.30Å R-factor: 0.219 R-free: 0.247

Authors:

C.Neubauer,Y.-G.Gao,K.R.Andersen,C.M.Dunham,A.C.Kelley,J.Hentschel, K.Gerdes,V.Ramakrishnan,D.E.Brodersen

Key ref:

C.Neubauer et al. (2009). The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE. Cell, 139, 1084-1095. PubMed id: 20005802 DOI: 10.1016/j.cell.2009.11.015

Date:

02-Nov-09 Release date: 15-Dec-09

Protein chain

Q5SLP7  (RL1_THET8) -  50S ribosomal protein L1 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 229 a.a.

Struc: 120 a.a.

Protein chain

P60405  (RL2_THET8) -  50S ribosomal protein L2 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 276 a.a.

Struc: 271 a.a.

Protein chain

Q5SHN8  (RL3_THET8) -  50S ribosomal protein L3 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 206 a.a.

Struc: 204 a.a.

Protein chain

Q5SHN9  (RL4_THET8) -  50S ribosomal protein L4 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 210 a.a.

Struc: 207 a.a.

Protein chain

Q5SHQ0  (RL5_THET8) -  50S ribosomal protein L5 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 182 a.a.

Struc: 181 a.a.

Protein chain

Q5SHQ3  (RL6_THET8) -

Protein chain

Q5SLQ1  (RL9_THET8) -  50S ribosomal protein L9 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 148 a.a.

Struc: 145 a.a.

Protein chain

Q8VVE3  (RL10_THET8) -  50S ribosomal protein L10 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 173 a.a.

Struc: 130 a.a.

Protein chain

P60488  (RL13_THET8) -  50S ribosomal protein L13 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 140 a.a.

Struc: 138 a.a.

Protein chain

Q5SHP8  (RL14_THET8) -  50S ribosomal protein L14 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 122 a.a.

Struc: 122 a.a.

Protein chain

Q5SHQ7  (RL15_THET8) -  50S ribosomal protein L15 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 150 a.a.

Struc: 146 a.a.

Protein chain

P60489  (RL16_THET8) -  50S ribosomal protein L16 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 141 a.a.

Struc: 140 a.a.

Protein chain

Q9Z9H5  (RL17_THET8) -  50S ribosomal protein L17 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 118 a.a.

Struc: 117 a.a.

Protein chain

Q5SHQ4  (RL18_THET8) -  50S ribosomal protein L18 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 112 a.a.

Struc: 98 a.a.

Protein chain

P60490  (RL19_THET8) -  50S ribosomal protein L19 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 146 a.a.

Struc: 135 a.a.

Protein chain

P60491  (RL20_THET8) -  50S ribosomal protein L20 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 118 a.a.

Struc: 117 a.a.

Protein chain

P60492  (RL21_THET8) -  50S ribosomal protein L21 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 101 a.a.

Struc: 101 a.a.

Protein chain

Q5SHP3  (RL22_THET8) -  50S ribosomal protein L22 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 113 a.a.

Struc: 113 a.a.

Protein chain

Q5SHP0  (RL23_THET8) -  50S ribosomal protein L23 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 96 a.a.

Struc: 92 a.a.

Protein chain

Q5SHP9  (RL24_THET8) -  50S ribosomal protein L24 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 110 a.a.

Struc: 100 a.a.

Protein chain

Q5SHZ1  (RL25_THET8) -  50S ribosomal protein L25 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 206 a.a.

Struc: 184 a.a.

Protein chain

P60493  (RL27_THET8) -  50S ribosomal protein L27 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 85 a.a.

Struc: 84 a.a.

Protein chain

P60494  (RL28_THET8) -  50S ribosomal protein L28 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 98 a.a.

Struc: 93 a.a.

Protein chain

Q5SHP6  (RL29_THET8) -  50S ribosomal protein L29 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 72 a.a.

Struc: 71 a.a.

Protein chain

Q5SHQ6  (RL30_THET8) -  50S ribosomal protein L30 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 60 a.a.

Struc: 59 a.a.

Protein chain

Q5SJE1  (RL31_THET8) -  50S ribosomal protein L31 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 71 a.a.

Struc: 57 a.a.

Protein chain

P80339  (RL32_THET8) -  50S ribosomal protein L32 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 60 a.a.

Struc: 55 a.a.

Protein chain

P35871  (RL33_THET8) -  50S ribosomal protein L33 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 54 a.a.

Struc: 50 a.a.

Protein chain

P80340  (RL34_THET8) -  50S ribosomal protein L34 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 49 a.a.

Struc: 47 a.a.

Protein chain

Q5SKU1  (RL35_THET8) -  50S ribosomal protein L35 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 65 a.a.

Struc: 63 a.a.

Protein chain

Q5SHR2  (RL36_THET8) -  50S ribosomal protein L36 from Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)

Seq: 37 a.a.

Struc: 37 a.a.

DNA/RNA chains

A-G-A-U-G-G-U-A-A-G-G-G-C-C-C-A-C-G-G-U-G-G-A-U-G-C-C-U-C-G-G-C-A-C-C-C-G-A-G- ... 2848 bases

U-C-C-C-C-C-G-U-G-C-C-C-A-U-A-G-C-G-G-C-G-U-G-G-A-A-C-C-A-C-C-C-G-U-U-C-C-C-A- 119 bases

DOI no: 10.1016/j.cell.2009.11.015

Cell 139:1084-1095 (2009)

PubMed id: 20005802

The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE.

C.Neubauer, Y.G.Gao, K.R.Andersen, C.M.Dunham, A.C.Kelley, J.Hentschel, K.Gerdes, V.Ramakrishnan, D.E.Brodersen.

ABSTRACT

Translational control is widely used to adjust gene expression levels. During the stringent response in bacteria, mRNA is degraded on the ribosome by the ribosome-dependent endonuclease, RelE. The molecular basis for recognition of the ribosome and mRNA by RelE and the mechanism of cleavage are unknown. Here, we present crystal structures of E. coli RelE in isolation (2.5 A) and bound to programmed Thermus thermophilus 70S ribosomes before (3.3 A) and after (3.6 A) cleavage. RelE occupies the A site and causes cleavage of mRNA after the second nucleotide of the codon by reorienting and activating the mRNA for 2'-OH-induced hydrolysis. Stacking of A site codon bases with conserved residues in RelE and 16S rRNA explains the requirement for the ribosome in catalysis and the subtle sequence specificity of the reaction. These structures provide detailed insight into the translational regulation on the bacterial ribosome by mRNA cleavage.

Selected figure(s)

Figure 1.
Figure 1. Overview of the RelE-Bound 70S Ribosome
(A) Top view of the 70S ribosome with the 50S (blue) and 30S (wheat) subunits surrounding RelE (A site, blue), tRNA^fMet (P site, green), a noncognate tRNA^fMet (E site, red), and mRNA (magenta). (A)–(C) are based on the precleavage structure.
(B) Close-up of the A and P sites of the 30S subunit viewed from the interface to the 50S. RelE (blue cartoon) spans the 16S rRNA from the head (helix 31 region, green) to the body (helix 18, pink). The mRNA is shown in purple sticks, and the P and E site tRNAs colored as in (A).
(C) Close-up view of the A and P sites showing RelE (blue Cα trace), mRNA (purple sticks), and P site tRNA (green cartoon) along with the DF[o]-mF[c] electron density of the precleavage structure contoured at 1.5 σ. The mRNA sequence is indicated.
(D) The postcleavage structure showing the position of the 2′-3′ cyclic phosphate generated upon cleavage (2′-3′ cP). The map is contoured at 1.2 σ.
See also Figure S1.

Figure 3.
Figure 3. In Vitro mRNA Cleavage Assay on the 70S Ribosome
(A) Sequence of E. coli RelE with the conservation among homologs indicated as increasing strength of red color and the conserved tyrosine at the C terminus in light blue. The secondary structure is shown above the sequence and the interactions to rRNA and mRNA below (all numbers correspond to the E. coli 16S sequence). Residues in the P. horikoshii RelE homolog that affect the activity are indicated with black boxes (Takagi et al., 2005).
(B) Overview of the mRNAs used for the in vitro cleavage assays. The 25 nt mRNA consists of a Shine-Dalgarno element (SD) followed by a spacer and the P site (AUG) and A site (UAG) codons. “Trunc Asite” ends after the P site codon with a 3′-OH. The table shows predicted masses of full length mRNA and fragments that would result from cleavage after position 1 or 2 of the A site codon leaving either a 3′-OH, 3′-phosphate (3′-P), or 2′-3′ cyclic phosphate (2′,3′-cP).
(C) MALDI mass spectrometry spectra and masses of RNA fragments isolated from complexes in the absence (blue) or presence (red) of RelE.
(D) In vitro cleavage assay using 5′ ^32P-labeled mRNA substrates. • is the 25 nt unmodified mRNA; MAP has phosphorothioate linkages after A site codon positions 1 and 2; MAO, MAO2, and MAO3 are 2′-O-methylated at position 1, positions 1 + 2, or all three positions, respectively; and MAD contains a deoxyribose at position 1. The mRNAs were incubated with either T. thermophilus (T.th.) or E. coli (E.co.) 70S ribosomes, tRNA^fMet, and either RelE^wt or RelE^R81A/R45A (RelE^dm) as indicated for either 1 hr (lanes 1–16) or overnight (lanes 17 and 18). The size markers indicate the positions of the full-length (25 nt 3′-OH) and Trunc Asite (18 nt 3′-OH) RNAs and the 20 nt 2′-3′ cyclic phosphate cleavage product, which runs approximately 1 nt faster than the corresponding 3′-OH species.
See also Figure S3.

The above figures are reprinted by permission from Cell Press: Cell (2009, 139, 1084-1095) copyright 2009.

Figures were selected by the author.