Molecular Biochemistry II

tRNA & Ribosomes

Copyright © 1999-2008 by Joyce J. Diwan. All rights reserved.

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

Purines & Pyrimidines

Nucleic acids

are polymers of

nucleotides

.

Each nucleotide includes a

base

that is either  a

purine

(adenine or guanine), or  a

pyrimidine

(cytosine, uracil, or thymine).

N NH 2 N

Nucleoside bases found in RNA:

O NH 2 N HN N O NH N N H H 2 N N N H N H O N H adenine (A) guanine (G) cytosine (C) uracil (U) O H 3 C + N NH 2 N HN O + N CH 3 NH 2 + N CH 3 HN O NH N N H H 2 N N N H N H O N H O 1-methyladenine (m 1 A) 7-methylguanine (m 7 G) 3-methylcytosine (m 3 C) pseudouracil (  )

Some nucleic acids contain modified bases. Examples: N NH 2 N

Nucleoside bases found in RNA:

O NH 2 N HN N O NH H 3 C + N N N H H 2 N N N H N H O N H adenine (A) guanine (G) cytosine (C) uracil (U) O NH 2 N

Examples of modified bases found in tRNA:

O CH 3 NH 2 + N CH 3 HN + N HN O NH N N H H 2 N N N H N H O N H O 1-methyladenine (m 1 A) 7-methylguanine (m 7 G) 3-methylcytosine (m 3 C) pseudouracil (  )

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 NH 2 N N NH 2 adenine N N NH 2 N N N H N N HO 5' CH 2 ribose 4' H H 3' OH O H 2' H OH 1'  2 O 3 P O CH 2 H N H OH O H N OH H adenine adenosine adenosine monophosphate (AMP)

Nucleic acids

have a backbone of alternating P i ribose moieties.

&

Phosphodiester

linkages form as the 5' phosphate of one nucleotide forms an ester link with the 3' OH of the adjacent nucleotide.

A short stretch of RNA is shown.

nucleic acid N NH 2 adenine N 5' end  O O  P O N N O  O 5' CH 2 4' H H O 3' O H 2' H OH 1' ribose P O 5' CH 2 O O H  O H O 3' P O NH 2 cytosine N N H OH H ribose O (etc) 3' end O

H cytosine (C) H N N guanine (G) N N H O N N H N H H O NH

G C G



C

base pair in tRNA

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 tRNA Gln . H atoms are not shown.

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 above and below, in the ladder-like arrangement of base pairs in the double helix, e.g., of DNA.

Genetic code

The

genetic code

is based on the sequence of bases along a nucleic acid. Each

codon

, a sequence of

3 bases

in mRNA, codes for a 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) in most cases differ only in the 3 rd base. Similar codons tend to code for similar amino acids. Thus effects of mutation are minimized.

1 st base U U

UUU Phe UUC Phe UUA Leu

C A

UUG Leu CUU Leu CUC Leu CUA Leu CUG Leu AUU Ile AUC Ile AUA Ile

G

AUG Met* GUU Val GUC Val GUA Val GUG Val *Met and initiation.

Genetic Code 2 nd base C A

UCU Ser UCC Ser UCA Ser UAU Tyr UAC Tyr UAA Stop UCG Ser CCU Pro CCC Pro CCA Pro CCG Pro ACU Thr ACC Thr ACA Thr ACG Thr GCU Ala GCC Ala GCA Ala GCG Ala UAG Stop CAU His CAC His CAA Gln CAG Gln AAU Asn AAC Asn AAA Lys AAG Lys GAU Asp GAC Asp GAA Glu GAG Glu

G

UGU Cys UGC Cys UGA Stop UGG Trp CGU Arg CGC Arg CGA Arg CGG Arg AGU Ser AGC Ser AGA Arg AGG Arg GGU Gly GGC Gly GGA Gly GGG Gly

3 rd base U C A G U C A G U C A G U C A G

tRNA

The genetic code is read during translation via adapter molecules,

tRNAs

, that have 3-base

anticodons

complementary to codons in mRNA.

"

Wobble

" during reading of the mRNA allows some tRNAs 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

.

RNA structure:

Most RNA molecules have

secondary structure

, consisting of

stem

&

loop

domains.

A U A C C

: : : : :

U A U G G G C U U U stem loop 

Double helical stem domains

arise from

base pairing

between complementary stretches of bases within the same strand.

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



Loop domains

occur where

lack of complementarity

or the presence of

modified bases

prevents base pairing.

anticodon loop tRNA acceptor stem The “

cloverleaf

” model of

tRNA

emphasizes the two major types of secondary structure,

stems & loops

. tRNAs typically include many

modified bases

, particularly in loop domains.

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.

anticodon loop Extending from the

acceptor stem

, the

3' end

of each tRNA has the sequence

CCA

.

tRNA acceptor stem The appropriate

amino acid

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 tRNA Phe acceptor stem

#22 G #46 (m 7 G) #13 C #22 G #46 (m 7 G) #13 C Tertiary base pairs in tRNA Phe Tertiary base pairs in tRNA Phe An example of

non-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).

Some other RNAs, including viral RNAs & segments of ribosomal RNA, fold in

pseudoknots

, tertiary structures that mimic the 3D structure of tRNA. Pseudoknots are similarly stabilized by non-standard H-bond interactions.

Explore

tRNA Phe with Chime (PDB file 1TRA) .

anticodon tRNA Phe acceptor stem

O R H C C NH 3 + Amino acid O    O O O R H C NH 2 C O Aminoacyl

-

AMP O P O O P O  ATP O  P O  O CH 2 H H OH O O O P O  O Adenine H OH H CH 2 H H OH O  PP i Adenine H OH H

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.

In

step 2

, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid carbonyl C atom.

O O R H C C O NH 2 Aminoacyl

-

AMP P O  O  CH 2 H H OH O tRNA AMP Adenine H OH H O tRNA O P O  O CH 2 H O H O 3’ 2’ OH H C O HC NH 3 + R H Adenine (terminal 3 ’ nucleotide of appropriate tRNA) Aminoacyl -tRNA

Aminoacyl-tRNA Synthetase

- summary: 1.

amino acid + ATP



aminoacyl -AMP + PP i

2.

aminoacyl -AMP + tRNA



aminoacyl -tRNA + AMP

The 2-step reaction is

spontaneous

overall, because the concentration of

PP i

is kept low by its hydrolysis, catalyzed by Pyrophosphatase.

There is generally a different

Aminoacyl-tRNA Synthetase

(

aaRS

) for each amino acid. Accurate translation of the genetic code depends on attachment of each amino acid to an appropriate tRNA.

Each

aaRS recognizes

its particular

amino acid

coding for that amino acid.

&

tRNAs 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 tRNA acceptor stem

There are 2 families of Aminoacyl-tRNA Synthetases:

Class I & Class II

. O tRNA O P O  O (terminal 3 ’ nucleotide of appropriate tRNA) CH 2 O Adenine H H H O 3’ 2’ OH H C O HC NH 3 + R Aminoacyl -tRNA 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').

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.

Proofreading/quality control:

Some Aminoacyl-tRNA Synthetases are known to have separate catalytic sites that

release by hydrolysis inappropriate amino acids

transferred to tRNA. that are misacylated or mis E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a small percentage of the time activates the closely related amino acid valine to valine-AMP. After

valine

is transferred to tRNA Ile , to form Val-tRNA Ile , 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.

Some amino acids are

modified

tRNA. Examples: after being linked to a  In prokaryotes the initiator

tRNA fMet

is first charged with

methionine

. Methionyl-tRNA formyltransferase then catalyzes formylation of the methionine, using

tetrahydrofolate

as formyl donor, to yield

formylmethionyl-tRNA fMet

.  In some prokaryotes, a

non-discriminating aaRS loads aspartate

onto

tRNA Asn

. The aspartate moiety is then

converted

by an amido transferase

to asparagine

, yielding Asn-tRNA Asn .

Glu-tRNA Gln Gln-tRNA Gln

is similarly formed and converted to in such organisms.

 Some proteins contain the unusual amino acid

selenocysteine

(

Sec

), with selenium substituting for the S atom in cysteine. H H H 3 N + C COO  H 3 N + C COO  CH 2 CH 2 SH SeH cysteine selenocysteine There is a

selenocysteine tRNA

that differs from other tRNAs, e.g., in having a slightly longer acceptor stem & a unique modified base in the anticodon loop.

tRNA Sec

is

loaded with serine

via Seryl-tRNA Synthetase. The

serine

moiety is then

converted to selenocysteine

by another enzyme, in a reaction involving selenophosphate.

Sec-tRNA Sec

utilization during protein synthesis requires special

elongation factors

because the codon for selenocysteine is

UGA

, which normally is a stop codon.

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

or

intron splicing

through binding to DNA or RNA.

 Proteolytic

cleavage

of the human

aaRS Tyr cytokine

that stimulates

angiogenesis

. yields a A

truncated

form of the human

aaRS Trp

angiogenesis. inhibits  Regulation of

apoptosis

by the human

aaRS Gln

dependent on the concentration of its substrate glutamine.

is  Several mammalian Aminoacyl-tRNA Synthetases associate with other proteins to form large

macromolecular complexes

being investigated.

whose roles are actively

Ribosome Composition

(S = sedimentation coefficient) Ribosome Source E. coli Rat cytoplasm Whole Ribosome 70S 80S Small Subunit 30S 16S RNA 21 proteins 40S 18S RNA 33 proteins Large Subunit 50S 23S & 5S RNAs 31 proteins 60S 28S, 5.8S, &5S RNAs 49 proteins Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. Mitochondrial and chloroplast ribosomes differ from both examples shown.

5S rRNA “crown” view displayed as ribbons & sticks. PDB 1FFK eukaryotic ribosomes have been determined, by X-ray crystallography & by cryo-EM with image reconstruction. Consistent with predicted base pairing, X-ray crystal structures indicate that

ribosomal RNAs

(rRNAs) have extensive

secondary structure

.

Structure of the

E. coli

Ribosome

large subunit tRNA EF-G mRNA location small subunit The cutaway view at right shows positions of

tRNA

(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 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.

Small ribosomal subunit

of a thermophilic bacterium:

rRNA

in monochrome;

proteins

in varied colors.

30S ribosomal subunit PDB 1FJF spacefill display ribbons The overall

shape

of the 30S ribosomal subunit is largely determined by the

rRNA

. The rRNA mainly consists of double 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.

Large ribosome subunit:

The

interior

of the large subunit is mostly

RNA

.

Proteins

are distributed mainly on the

surface

.

Some proteins have long tails that extend into the interior of the complex. These tails, which are highly

basic

, interact with the negatively charged RNA. PDB 1FFK Large Ribosome Subunit "Crown" view with RNAs blue, in spacefill; proteins red, as backbone.

The

active site

domain for peptide bond formation is essentially devoid of protein. The

peptidyl transferase

function is attributed to

23S rRNA

, making this RNA a "

ribozyme

." PDB 1FFK Large Ribosome Subunit "Crown" view with RNAs blue, in spacefill; proteins red, as backbone.

Protein synthesis

takes place in a

cavity

within the ribosome, between small & large subunits. Nascent polypeptides emerge through a

tunnel

in the large subunit. The tunnel lumen is lined with rRNA helices and some ribosomal proteins.

PDB 1FFK Large ribosome subunit. Backbone display with RNAs blue. View from bottom at tunnel exit.

Catalysis of

protein synthesis

and movement of the ribosome relative to messenger RNA are accompanied by

changes in ribosome conformation

.

EM

&

X-ray

crystallographic studies, carried out in the presence & absence of initiation & elongation factors as well as inhibitors of protein synthesis, have revealed

conformational changes in rRNA

. Thus

rRNA

participates in

conformational coupling

addition to its structural & catalytic roles. in

tRNAs

also undergo substantial

conformational changes

within ribosomal binding sites during protein synthesis.

Explore

the large ribosomal subunit.