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tRNA & Ribosomes

Copyright 1999-2005 by Joyce J. Diwan.

All rights reserved.

Molecular Biochemistry II


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Molecular Biology

Familiarity with basic concepts is assumed, including:

nature of the genetic code

maintenance of genes through DNA replication

transcription of information from DNA to mRNA

translation of mRNA into protein.

DNA mRNA protein


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Purines & Pyrimidines

N

NNH

N

NH2

HN

NNH

N

O

H2N

N

NH

NH2

O

NH

NH

O

O

adenine (A) guanine (G) cytosine (C) uracil (U)

Nucleoside bases found in RNA:

Nucleic acids are polymers ofnucleotides.

Each nucleotide includes a base that is either

a purine (adenine or guanine), or

a pyrimidine (cytosine, uracil, or thymine).


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Some nucleic acids contain modified bases. Examples:

N

NNH

N

NH2

HN

NNH

N

O

H2N

N

N

H

NH2

O

NH

NH

O

O

adenine (A) guanine (G) cytosine (C) uracil (U)

N

NNH

N

NH2

H3C

+HN

NNH

N

O

H2N

CH3

+

N

NH

NH2

O

CH3+NH

NH

HN

O

O

1-methyladenine (m1A) 7-methylguanine (m7G) 3-methylcytosine (m3C) pseudouracil ()

Nucleoside bases found in RNA:

Examples of modified bases found in tRNA:


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In a nucleotide, e.g., adenosine monophosphate (AMP),

the base is bonded to a ribose sugar, which has a phosphate

in ester linkage to the 5' hydroxyl.

N

NN

N

NH2

adenine adenosine adenosine monophosphate (AMP)

O

OHOH

HH

H

CH2

H

HO

N

NNH

N

NH2

N

NN

N

NH2

O

OHOH

HH

H

CH2

H

OO3P2

ribose

5'

adenine

4'

3' 2'

1'


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Nucleic acids

have a backbone

of alternating Pi &ribose moieties.

Phosphodiester

linkages form as

the 5' phosphateof one nucleotide

forms an ester link

with the 3' OH of

the adjacent

nucleotide.

A short stretch of

RNA is shown.

N

NN

N

NH2

O

OHO

HH

H

CH2

H

ribose

adenine

P

O

O OO

OHO

HH

H

CH2

H

N

N

NH2

O

P

O

O O

OP

O

O

O

cytosine5'

4'

3' 2'

1'

ribose3'

5'

3'end

5'end

(etc)

nucleic acid


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Hydrogen bonds link 2 complementary nucleotide bases

on separate nucleic acid strands, or on complementary

portions of the same strand.Conventional base pairs: A & U (or T); C & G.

In the diagram at left, H-bonds are in red. Bond lengths

are inexact. The image at right is based on X-ray

crystallography of tRNAGln. H atoms are not shown.

N

N

NH

N

O

N

N

NH

N

O

H

H

H

H

H

guanine(G)

cytosine(C)

G

C

GC basepair in tRNA


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Secondary structure

Base pairing over extended stretches of complementary

base sequences in two nucleic acid strands stabilizes

secondary structure, such as the double helix of DNA.

Stacking interactions between adjacent hydrophobic

bases contribute to stabilization of such secondary

structures. Each base interacts with its neighbors aboveand below, in the ladder-like arrangement of base pairs

in the double helix, e.g., of DNA.


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Genetic code

The genetic code is based on the sequence of bases along

a nucleic acid.

Each codon, a sequence of3 bases in mRNA, codes fora particular amino acid, or for chain termination.

Some amino acids are specified by 2 or more codons.

Synonyms (multiple codons for the same amino acid) inmost cases differ only in the 3rd base. Similar codons

tend to code for similar amino acids. Thus effects of

mutation are minimized.


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Genetic Code

2nd

base1st

base

U C A G

3rd

base

UUU Phe UCU Ser UAU Tyr UGU Cys U

UUC Phe UCC Ser UAC Tyr UGC Cys CUUA Leu UCA Ser UAA Stop UGA Stop A

U

UUG Leu UCG Ser UAG Stop UGG Trp G

CUU Leu CCU Pro CAU His CGU Arg U

CUC Leu CCC Pro CAC His CGC Arg C

CUA Leu CCA Pro CAA Gln CGA Arg A

C

CUG Leu CCG Pro CAG Gln CGG Arg G

AUU Ile ACU Thr AAU Asn AGU Ser U

AUC Ile ACC Thr AAC Asn AGC Ser C

AUA Ile ACA Thr AAA Lys AGA Arg A

A

AUG Met* ACG Thr AAG Lys AGG Arg G

GUU Val GCU Ala GAU Asp GGU Gly U

GUC Val GCC Ala GAC Asp GGC Gly C

GUA Val GCA Ala GAA Glu GGA Gly A

G

GUG Val GCG Ala GAG Glu GGG Gly G*Met and initiation.


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tRNA

The genetic code is read during translation via adaptermolecules, tRNAs, that have 3-base anticodons

complementary to codons in mRNA.

"Wobble" during reading of the mRNA allows sometRNAs to read multiple codons that differ only in the

3rd base.

There are 61 codons specifying 20 amino acids.

Minimally 31 tRNAs are required for translation, not

counting the tRNA that codes for chain initiation.

Mammalian cells produce more than 150 tRNAs.


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Double helical stem domains arise from base pairing

between complementary stretches of bases within the

same strand.

These stem structures are stabilized by stackinginteractions as well as base pairing, as in DNA.

Loop domains occur where lack of complementarity

or the presence ofmodified bases prevents base pairing.

A U A C C

U A U G G

C U

C U

G

U

U

stem loop

: : : : :

RNAstructure:

Most RNA molecules havesecondary structure,consisting ofstem & loop

domains.


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The cloverleaf model oftRNA emphasizes the two

major types of secondary structure, stems & loops.tRNAs typically include many modified bases,

particularly in loop domains.

anticodon loop

acceptor

stemtRNA


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RNA tertiary structure depends on interactions of

bases at distant sites.

These interactions generally involve non-standard

base pairing and/or interactions involving three or

more bases.

Unpaired adenosines (not involved in conventional

base pairing) predominate in participating in non-

standard interactions that stabilize tertiary RNA

structures.

tRNAs have an L-shaped tertiary structure.


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The appropriate aminoacid is attached to the

ribose of the terminal

adenosine (A, in red) at

the 3' end.

The anticodon loop is

at the opposite end of

the L shape.

anticodon

acceptor

stem

tRNAPhe

anticodon loop

acceptorstemtRNA

Extending from the acceptor stem,

the 3' end of each tRNA has the

sequence CCA.


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#46(m7G)

#22

G #13

C

Tertiary base

pairs in tRNAPhe

#46(m

7G)

#22

G

#13

C

Tertiary base

pairs in tRNAPhe

An example ofnon-standard H bond interactions that

help to stabilize the L-shaped tertiary structure of a tRNA,

in ball & stick & spacefill displays.

H atoms are not shown. (From NDB file 1TN2).


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Some other RNAs,

including viral RNAs &

segments of ribosomalRNA, fold in

pseudoknots, tertiary

structures that mimic the

3D structure of tRNA.

Pseudoknots are

similarly stabilized by

non-standard H-bondinteractions.

Explore tRNAPhe with

Chime(PDB file 1TRA)

.

anticodon

acceptorstem

tRNAPhe
http://localhost/var/www/apps/conversion/tmp/scratch_8/trna.htmhttp://localhost/var/www/apps/conversion/tmp/scratch_8/trna.htm


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Aminoacyl-tRNA Synthetases catalyze linkage of the

appropriate amino acid to each tRNA.

The reaction occurs in 2 steps.

In step 1, an O atom of the amino acid a-carboxyl attacks

the P atom of the initial phosphate of ATP.

O

OHOH

HH

H

CH2

H

OPOPOPO

O

O

O

O O

O

R

H

C C

NH3+

O

O

O

OHOH

HH

H

CH2

H

OPOC

O

O

H

CR

NH2

O

Adenine

Adenine

ATPAmino acid

Aminoacyl-AMP

PPi


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In step 2, the

2' or 3' OH of

the terminal

adenosine of

tRNA attacks

the amino acidcarbonyl C

atom.

O

OHOH

HH

H

CH2

H

OPOC

O

O

H

CR

NH2

O

Adenine

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

O

NH3+

R

tRNA

AMP

Aminoacyl-AMP

Aminoacyl-tRNA

(terminal 3nucleotide

of appropriate tRNA)3 2


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Aminoacyl-tRNA Synthetase - summary:

1. amino acid+ ATPaminoacyl-AMP + PPi

2. aminoacyl-AMP+tRNAaminoacyl-tRNA+ AMP

The 2-step reaction is spontaneous overall, because the

concentration ofPPi is kept low by its hydrolysis,

catalyzed by Pyrophosphatase.


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There is generally a different Aminoacyl-tRNA

Synthetase (aaRS) for each amino acid.

Accurate translation of the genetic code depends onattachment of each amino acid to an appropriate tRNA.

Each aaRS recognizes its particularamino acid & tRNAs

coding for that amino acid.

Identity elements: tRNA domains recognized by an aaRS.

Most identity elements are in the

acceptor stem & anticodon loop.

Aminoacyl-tRNA Synthetases arose

early in evolution.

Early aaRSs probably recognized

tRNAs only by their acceptor stems.

anticodon loop

acceptor

stemtRNA


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Two different ancestral proteins evolved into the 2 classes

of aaRS enzymes, which differ in the architecture of their

active site domains.

They bind to opposite sides of the tRNA acceptor stem,

aminoacylating a different OH of the tRNA (2' or 3').

O

OHO

HH

H

CH2

H

OPO

O

O

Adenine

tRNA

C

HC

O

NH3+

R

Aminoacyl-tRNA

(terminal 3nucleotide

of appropriate tRNA)

3 2

There are 2 families

of Aminoacyl-tRNASynthetases:

Class I & Class II.


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Class I aaRSs:

Identity elements usually include residues of the

anticodon loop & acceptor stem.

Class I aaRSs aminoacylate the 2'-OH of adenosine at

their 3' end.

Class II aaRSs:

Identity elements for some Class II enzymes do not

include the anticodon domain.

Class II aaRSs tend to aminoacylate the 3'-OH of

adenosine at their 3' end.


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Proofreading/quality control:

Some Aminoacyl-tRNA Synthetases are known to have

separate catalytic sites that release by hydrolysisinappropriate amino acids that are misacylated or mis-

transferred to tRNA.

E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a

small percentage of the time activates the closely relatedamino acid valine to valine-AMP.

Aftervaline is transferred to tRNAIle, to form Val-tRNAIle,

it is removed by hydrolysis at a separate active site of

IleRS that accommodates Val but not the larger Ile.

In some bacteria, editing of some misacylated tRNAs is

carried out by separate proteins that may be evolutionary

precursors to editing domains of aa-tRNA Synthetases.


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Some amino acids are modified after being linked to a

tRNA. Examples:

In prokaryotes the initiatortRNAfMet

is first chargedwith methionine.

Methionyl-tRNA formyltransferase then catalyzes

formylation of the methionine, using tetrahydrofolate

as formyl donor, to yield formylmethionyl-tRNAfMet.

In some prokaryotes, a non-discriminating aaRS

loadsaspartate onto tRNAAsn.

The aspartate moiety is then converted by an amido-transferase to asparagine, yielding Asn-tRNAAsn.

Glu-tRNAGln is similarly formed and converted to

Gln-tRNAGln in such organisms.


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Some proteins contain the unusual amino acidselenocysteine (Sec), with selenium substituting for

the sulfur atom in cysteine.

ThetRNASec

is first loaded with serine via Seryl-tRNASynthetase.

The serine moiety is then converted to selenocysteine

by another enzyme.

Utilization of Sec-tRNASec during protein synthesis also

requires special elongation factors.


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Other roles of aminoacyl-tRNA synthetases:

In some organisms, Aminoacyl-tRNA Synthetases

(aaRSs) have evolved to take on signaling roles in

addition to the catalytic role of joining an amino acid

to the correct tRNA.

Examples have been identified of particular aaRSs

that regulate transcription, translation orintron

splicing through binding to DNA or RNA.


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Proteolytic cleavage of the human aaRSTyr yields a

cytokine that stimulates angiogenesis.

A truncated form of the human aaRSTrp inhibits

angiogenesis.

Regulation ofapoptosis by the human aaRSGln isdependent on the concentration of its substrate

glutamine.

Several mammalian Aminoacyl-tRNA Synthetasesassociate with other proteins to form large

macromolecular complexes whose roles are actively

being investigated.


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Eukaryotic cytoplasmic ribosomes are larger and more

complex than prokaryotic ribosomes. Mitochondrial and

chloroplast ribosomes differ from both examples shown.

Ribosome

Source

Whole

Ribosome

Small

Subunit

Large

Subunit

E. coli 70S 30S

16S RNA

21 proteins

50S

23S & 5S

RNAs

31 proteinsRat

cytoplasm

80S 40S

18S RNA

33 proteins

60S

28S, 5.8S, &5S

RNAs

49 proteins

Ribosome Composition (S = sedimentation coefficient)


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Structures of large & small subunits of bacterial &

eukaryotic ribosomes have been determined, by X-raycrystallography & by cryo-EM with image reconstruction.

Consistent with predicted base pairing, X-ray crystal

structures indicate that ribosomal RNAs (rRNAs) have

extensive secondary structure.

5S rRNA

crown view

displayed asribbons & sticks. PDB 1FFK


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Structure of theE. coliRibosome

The cutaway view at right shows positions oftRNA (P, E

sites) & mRNA (as orange beads). EF-G will be discussed

later. This figure was provided by Joachim Frank, whose

lab at the Wadsworth Center carried out the cryo-EM and

3D image reconstruction on which the images are based.

small subunit

large subunit

mRNA

location

EF-G

tRNA


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Small Ribosomal Subunit

In the translation complex, mRNA threads through a

tunnel in the small ribosomal subunit.

tRNA binding sites are in a cleft in the small subunit.

The 3' end of the 16S rRNA of the bacterial small

subunit is involved in mRNA binding.

The small ribosomal subunit is relatively flexible,

assuming different conformations.E.g., the 30S subunit of a bacterial ribosome was

found to undergo specific conformational changes

when interacting with a translation initiation factor.

30S ib l b it PDB 1FJF


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The overall shape of the 30S ribosomal subunit is largely

determined by the rRNA. The rRNA mainly consists ofdouble helices (stems) connected by single-stranded loops.

The proteins generally have globular domains, as well as

long extensions that interact with rRNA and may stabilize

interactions between RNA helices.

30S ribosomal subunit PDB 1FJFSmall

ribosomal

subunit of a

thermophilicbacterium:

rRNA in

monochrome;

proteins invaried colors. spacefill display ribbons


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Large ribosome subunit:

The interior of the large

subunit is mostly RNA.

Proteins are distributed

mainly on the surface.

Some proteins have longtails that extend into the

interior of the complex.

These tails, which arehighly basic, interact

with the negatively

charged RNA.

PDB 1FFK

LargeRibosomeSubunit

"Crown" view with RNAs blue, ins acefill roteins red as backbone.


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The active site domain

for peptide bond

formation is essentiallydevoid of protein.

Peptidyl transferase is

attributed to 23S rRNA,making this RNA a

"ribozyme."

A universally conserved

adenosine base serves asa general acid base

during peptide bond

formation.

PDB 1FFK

Large

RibosomeSubunit

"Crown" view with RNAs blue, ins acefill roteins red as backbone.


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Protein synthesis takes

place in a cavity within

the ribosome.

Nascent polypeptides

emerge through a

tunnel in the large

subunit.Some nascent proteins

then pass through a

channel into the ER

lumen, or across the

cytoplasmic membrane

and out of the cell in

prokaryotes.

Large ribosome subunit.

Backbone display with RNAs blue. View

from bottom at tunnel exit.

PDB 1FFK


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The cutaway view at right shows that the tunnel in the

yeast large ribosome subunit, through which nascentpolypeptides emerge from the ribosome, lines up with the

lumen of the ERSec61 channel.

Figure provided by Joachim Frank, whose lab carried out the

EM & i t ti hi h th i b d

small

subunit large

subunit

Sec61 channel

path of

nascentprotein

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