Lecture PowerPoint to accompany

Molecular Biology
Fourth Edition

Robert F. Weaver
Chapter 19
Ribosomes and
Transfer RNA
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

19.1 Ribosomes
E. coli ribosome is a two-part structure
with a sedimentation coefficient of 70S
Two subunits of this structure:
30S is the small subunit that decodes mRNA
50S subunit links amino acids together
through peptide bonds

19-2

Fine Structure of 70S Ribosome

T. thermophilus crystal structure of 70S
ribosome in complex with mRNA analog and 3
tRNAs shows:
Positions and tertiary structures of all 3 rRNA and
most proteins can be determined
Shapes and locations of tRNAs in A, P, and E sites
are evident
Binding sites for tRNAs in ribosome are composed of
rRNA, not protein
Anticodons of tRNAs in A and P sites approach each
other closely enough to base-pair with adjacent
codons bound to 30S subunit as mRNA kinks 45

19-3

More Structural Detail

Acceptor stems of tRNAs in A and P sites
also approach each other closely (5 ) in
the peptidyl transferase pocket of 50S
subunit
Consistent with need for 2 stems to
interact during peptide bond formation
12 contacts are seen between subunits,
most RNA-RNA interactions
19-4

E. coli Ribosome Structure

Crystal structure of E. coli ribosome
contains 2 structures differing from each
other by rigid body motions of ribosome
domains relative to each other
Head of 30S particle rotates by 6
12 rotation compared to T. thermophilus
ribosome
Probably part of a ratchet action that occurs
during translocation
19-5

Eukaryotic Ribosomes
Eukaryotic cytoplasmic ribosomes are:
Larger
More complex

Eukaryotic organellar ribosomes are

smaller than prokaryotic ones

19-6

Ribosome Composition
The E. coli 30 subunit contains
16S rRNA
21 proteins (S1 S21)

E. coli 50S subunit contains

5S rRNA
23S rRNA
34 proteins (L1 L34)

Eukaryotic cytoplasmic ribosomes are:

Larger
Contain more RNAs and proteins

19-7

Ribosome Assembly
Assembly of the 30S ribosomal subunit in
vitro begins with 16S rRNA
Proteins join sequentially and
cooperatively
Proteins added early in the process
Help later proteins to bind to the growing
particle

19-8

Fine Structure of the 30S

Subunit
Sequence studies of 16S rRNA led to a
proposal for secondary structure of the
molecule
X-ray crystallography studies have
confirmed the conclusions of these studies
30S subunit with extensively base-paired 16S
rRNA whose shape essentially outlines the
whole particle
X-ray crystallography studies confirmed
locations of most of the 30S ribosomal
proteins
19-9

Interaction of the 30S Subunit

with Antibiotics
30S ribosomal subunit plays 2 roles
Facilitates proper decoding between codons
and aminoacyl-tRNA anticodons
Also participates in translocation

Crystal structures of 30S subunits with 3

antibiotics interferring with these 2 roles
shed light on translocation and decoding
Spectinomycin
Streptomycin
Paromomycin
19-10

Spectinomycin
Spectinomycin binds to 30S subunit near
the neck
At this site, binding interferes with
movement of the head
Head movement is required for
translocation

19-11

Streptomycin
Streptomycin binds near the A site of 30S
subunit
Binding stabilizes the ram state of the
ribosomes
Fidelity of translation is reduced:
Allowing noncognate aminoacyl-tRNAs to bind
easily to the A site
Preventing the shift to the restrictive state that
is necessary for proofreading
19-12

Paromomycin
Paromomycin binds in the minor groove of 16S
rRNA H44 helix near the A site
This binding flips out bases A1492 and A1493 to
stabilize base pairing between codon and anticodon
Flipping out process normally requires energy
Paromomycin forces it to occur and keeps the
stabilizing bases in place

State of the A site stabilizes codon-anticodon

interaction, including interaction between
noncognate codons and anticodons with decline
in fidelity
19-13

Interaction of the 30S Subunit

with Initiation Factors
X-ray crystal structure of IF1 bound to the 30S
ribosomal subunit shows IF1 binds to the A site
In that position IF1:
Blocks fMet-tRNA from binding to the A site
May also actively promote fMet-tRNA binding to P site
through interaction between IF1 and IF2

IF1 also interacts closely with helix H44 of the

30S subunit
IF accelerates both association and dissociation
of the ribosomal subunits
19-14

Fine Structure of the 50S

Subunit
Crystal structure of the 50S ribosomal
subunit has been determined to 2.4
Structure reveals relatively few proteins at
interface between ribosomal subunits
No proteins within 18 of peptidyl transferase
active center tagged with a transition state
analog
2-OH group of tRNA in the P site is very well
positioned to form a hydrogen bond to amino
group of aminoacyl-tRNA in A site
19-15

2-Hydroxyl Group Role

2-OH group of tRNA in the P site
Forms a hydrogen bond to amino group of aminoacyltRNA in A site
Helps catalyze peptidyl transferase reaction

Removal of this hydroxyl group eliminates

peptidyl transferase activity
Removal of the 2-OH of A2451 of the 23S rRNA
inhibits peptidyl transferase activity
May also participate in catalysis by:
Hydrogen bonding
Helping to position reactants properly for catalysis

19-16

50S Exit Tunnel

Exit tunnel through the 50S subunit
Just wide enough to allow a protein -helix to
pass
Walls of tunnel are made of RNA
Hydrophobicity is likely to allow exposed
hydrophobic side chains of nascent
polypeptide to slide through easily

19-17

Polysomes
Most mRNAs are translated by more than one
ribosome at at time
A structure in which many ribosomes translate
mRNA in tandem is called a polysome
Eukaryotic polysomes are found in the
cytoplasm
In prokaryotes, transcription of a gene and
translation of the resulting mRNA occur
simultaneously
Many polysomes are found associated with an
active gene
19-18

19.2 Transfer RNA

An adaptor molecule was proposed that
could serve as a mediator between string
of nucleotides in DNA or RNA and the
string of amino acids in the corresponding
protein
The adaptor contained 2 or 3 nucleotides
that could pair with nucleotides in codons

19-19

The Discovery of tRNA

Transfer RNA was discovered as a small
species independent of ribosomes
This small species could be charged with
an amino acid
That species could then pass the amino
acid to a growing polypeptide

19-20

tRNA Structure
All tRNAs share a common secondary
structure represented by a cloverleaf
Four base-paired stems define three
stem-loops
D loop
Anticodon loop
T loop

The acceptor stem is the site to which

amino acids are added in the charging
step
19-21

tRNA Shape
tRNAs share a common three-dimensional
shape resembling an inverted L
This shape maximizes stability by lining up
the base pairs:
In the D stem with those in the anticodon stem
In the T stem with those in the acceptor stem

Anticodon of the tRNA protrudes from the

side of the anticodon loop
Anticodon is twisted into a shape that basepairs with corresponding codon in mRNA
19-22

Modified Nucleosides in tRNA

19-23

Recognition of tRNA Acceptor

Stem
Biochemical and genetic experiments
have demonstrated the importance of the
acceptor stem in recognition of a tRNA by
its cognate aminoacyl-tRNA synthetase
Changing one base pair in the acceptor
stem can change the charging specificity

19-24

The Anticodon
Biochemical and genetic experiments
have shown that anticodon, like acceptor
stem, is an important element in charging
specificity
Sometimes the anticodon can be the
absolute determinant of specificity

19-25

Structures of Synthetase-tRNA
Complexes
Crystallography has shown that synthetasetRNA interactions differ between the 2
classes of aminoacyl-tRNA synthetases
Class I synthetases
Pockets for acceptor stem and anticodon of their
cognate tRNA
Approach the tRNAs from the D loop and acceptor
stem minor groove side

Class II synthetases
Also have pockets for acceptor stem and anticodon
Approach tRNA from opposite including the variable
arm and the major groove of the acceptor stem

19-26

Proofreading and Editing

Amino acid selectivity of at least some aminoacyltRNA synthetases is controlled by a double-sieve
mechanism
1st sieve is coarse excluding amino acids too big
Enzyme accomplishes this with an active site for
activation of amino acids just big enough to
accommodate the cognate amino acid, not larger amino
acids

2nd sieve is fine, degrades too small aminoacyl-AMPs

Done with a second active site, the editing site, admits
small aminoacyl-AMPs and hydrolyzes them
Cognate aminoacyl-AMP is too big to fit into the editing
site
Enzyme transfers the activated amino acid to its cognate
19-27
tRNA