Download Lecture Notes

Microreview
Nucleic acid structure
1er: sequence of the nucleotides, each distinguished by its
base.
2ary: antiparallel double helix.
3ary: folding of sequentially remote secondary structure
to form 3-d entity: coaxial stacks, pseudoknots,
ribose zippers, cation-binding sites.
4ary: association of multiple molecules to form a noncovalent complex, often in association with proteins.
A.-F. Miller, 2008, pg
1
Tertiary structure of RNA
Coaxial stacks of bases from the
bases of different stem-loops.
Pseudoknots: H-bonding between
loop bases and nearby SS-RNA.
Ribose zippers: H-bonds between
2’OH groups of ribose in
antiparallel SS strands.
Mg2+ binding, NEXT.
Three-base H-bonding.
11.34
A.-F.Fig.
Miller,
2008, pgof Garrett
2
& Grisham
Translation: tRNA and
Ribosomes
1-6 Strong sites: Kd~10-5 M,
cooperative.
Weak sites, statistical, ≈ ionic
strength effects.
Holbrook et al (1977) NAR 4(8) 2811.
A.-F. Miller, 2008, pg
3
Higher-order DNA
+
structure & K ions.
G-quartets in DNA
Kondo et al. (2004) NAR
32(8) 2541.
Different binding sites depending on
K+: DNA ratio.
Also: see Thermodynamics of RNA
folding vs. [Mg2+].
A.-F. Miller, 2008, pg
4
Quaternary structure of RNA
2er Str.
3er and 4er structure
50s subunit of ribosome including 5S rRNA and 23S rRNA.
RNA accounts for 65% of the mass of this particle.
A.-F. Miller, 2008, pg
5
Fig. 11.37 of Garrett & Grisham
Central dogma
Replication, transcription, translation (the simple version).
DNA
mRNA
rRNA, tRNA
Protein
Replication: DNA + dNTP →2 DNA
DNA polymerase (Mg2+)
Transcription: DNA + NTP →DNA + RNA
RNA polymerase (Mg2+)
Translation: RNA + aa →RNA + protein
ribosomes (Mg2+)
A.-F. Miller, 2008, pg
6
mRNA, tRNA, rRNA are involved
mRNA from prokaryotes vs. eukaryotes.
G&G Fig. 10.24
A.-F. Miller, 2008, pg
7
Transcription:
RNA
polymerase
Mg2+ and Zn2+
A.-F. Miller, 2008, pg
8
Fig3, Cramer (2000) Science 288:640
mRNA from prokaryotes vs. eukaryotes.
Protein synthesis video,
Stanford U. in the early 70s.
search ‘Paul Berg’ and ‘protein synthesis’.
A.-F. Miller, 2008, pg
9
G&G Fig. 10.24
The ribosome
MW 2.7 x 106 Da. (E. coli)
16S
Crystal structure of a Thermus
thermophilus 70S ribosome containing
three bound transfer RNAs (top) and
exploded views showing its different
molecular components (middle and
bottom). The 16S, 23S, and 5S ribosomal
RNAs are cyan, gray, and light purple,
respectively; the A-, P-, and E-site transfer
RNAs are shown in yellow, orange, and red,
respectively. The 30S subunit proteins are
dark purple, and the 50S proteins are
magenta.
A.-F. Miller, 2008, pg
10
5S
23S
A, P, E
30S
50S
The ribosome
A.-F. Miller, 2008, pg
11
Translation: tRNA and
Ribosomes
Acceptor, Peptidyl
and Exit sites.
tRNA molecules
serve as
adaptors basepairing with one
codon on the
mRNA and
bringing along
one amino acid
for the growing
peptide chain.
A.-F. Miller, 2008, pg
12
Figs. 30.19 of G&G
Formation of new peptide linkages
A.-F. Miller, 2008, pg
13
Figs. 30.21 of G&G
Charging of tRNA
Errors only 1 in 104-105
1 in 102-103x, Ser is adenylated, but it
is hydrolize at the acylation site.
A.-F. Miller, 2008, pg
14
Figs. 30.3 of G&G
Accuracy: For Thr vs. Val, Ser recognition,
a Zn2+ binds OH, not CH3 of Val.
Mg 2+ in the 30s
ribosomal
fragment
James M. Ogle, et al. (2001)
Science 292: pp 897-902 . “Recognition
of Cognate Transfer RNA by the 30
Ribosomal Subunit.”
A.-F. Miller, 2008, pg
15
70s Ribosomal fragment
(2006) Science 313 1935-1943
A.-F. Miller, 2008, pg
16
Mg2+ bridges 45° kink in
mRNA between P and A
sites, also binds bkbn
Phosphates of Helix44
residues 1400,1401.
3Fo-2Fc density is outlined.
All green balls are Mg2+.
Selmer (2006) Science 313 1935-1943.
A.-F. Miller, 2008, pg
17
Mg ions in 70s ribosomal
subunit.
→
In the 70s Ribosome, numerous Mg2+
ions are implicated in bridges between
ribosomal fragments. Interactions with
the RNA most commonly exploit
backbone phosphate (non-bridging O).
This is proposed to accommodate the
shifts in tRNA vs. mRNA and rRNA
and protein required as part of
ribosome function, as the tRNAs shift
from the ‘A’ site to the ‘P’ site.
Selmer (2006) Science 313 1935-1943.
A.-F. Miller, 2008, pg
18
Bridges
between
domains and
subunits.
C: Mg links the 2ʼ-OH of C770
and O2 of C899 of 16S, as
well as non-bridging
phosphate Os of 23S.
D: L14 protein and 16S and 23S
are bridged by Mg bound to side
chains of Glu and non-bridgin
phosphate Os.
E:L14 and L19 with 16S RNA,
backbone oxygens of both
Val115 and Ala118 of BUT:
“side chain of Lys35 of L19
and h14 of 16S RNA”
A.-F. Miller, 2008, pg
19
Selmer (2006) Science 313 1935-1943.