Biochemistry, 4th Edition P100 doc

To interpret this second genetic code, an aminoacyl-tRNA syn-thetase must discriminate between the 20 amino acids and the many tRNAs and uniquely picks out its proper substrates—one spec

3 The base sequence is read from a fixed starting point without punctuation That

is, the mRNA sequences contain no “commas” signifying appropriate groupings

of triplets If the reading frame is displaced by one base, it remains shifted

throughout the subsequent message; no “commas” are present to restore the

“correct” frame

4 The code is degenerate, meaning that, in most cases, each amino acid can be

coded by any of several triplets Recall that a triplet code yields 64 codons for

20 amino acids Most codons (61 of 64) code for some amino acid

Codons Specify Amino Acids

The complete translation of the genetic code is presented in Table 30.1 Codons,

like other nucleotide sequences, are read 5→3 Codons represent triplets of bases

in mRNA or, replacing U with T, triplets along the nontranscribed (nontemplate)

strand of DNA

Several noteworthy features characterize the genetic code:

1 All the codons have meaning Of the 64 codons, 61 specify particular amino acids.

The remaining 3—UAA, UAG, and UGA—specify no amino acid and thus they

are nonsense codons Nonsense codons serve as termination codons; they are

“stop” signals indicating that the end of the protein has been reached

2 The genetic code is unambiguous Each of the 61 “sense” codons encodes only one

amino acid

3 The genetic code is degenerate With the exception of Met and Trp, every amino acid

is coded by more than one codon Several—Arg, Leu, and Ser—are represented

by six different codons Codons coding for the same amino acid are called

synonymous codons.

4 Codons representing the same amino acid or chemically similar amino acids tend to be

simi-lar in sequence Often the third base in a codon is irrelevant, so, for example, all four

codons in the GGX family specify Gly, and the UCX family specifies Ser (Table

30.1) This feature is known as third-base degeneracy Note also that codons with a

pyrimidine as second base likely encode amino acids with hydrophobic side chains,

and codons with a purine in the second-base position typically specify polar or

charged amino acids The two negatively charged amino acids, Asp and Glu, are

encoded by GAX codons; GA–pyrimidine gives Asp and GA–purine specifies Glu

The consequence of these similarities is that mutations are less likely to be

harm-ful because single base changes in a codon will result either in no change or in a

substitution with an amino acid similar to the original amino acid The degeneracy

of the code is evolution’s buffer against mutational disruption

5 The genetic code is “universal.” Although certain minor exceptions in codon usage

occur (see A Deeper Look box on page 954), the more striking feature of the

code is its universality: Codon assignments are virtually the same throughout all

organisms—archaea, bacteria, and eukaryotes This conformity means that all

extant organisms use the same genetic code, providing strong evidence that they

all evolved from a common primordial ancestor

30.2 How Is an Amino Acid Matched with Its Proper tRNA?

Codon recognition is achieved by aminoacyl-tRNAs In order for accurate translation

to occur, the appropriate aminoacyl-tRNA must “read” the codon through base

pair-ing via its anticodon loop (see Chapter 11) Once an aminoacyl-tRNA has been

syn-thesized, the amino acid part makes no contribution to accurate translation of the

mRNA That is, the amino acid is passively chauffeured by its tRNA and becomes

in-serted into a growing peptide chain following codon–anticodon recognition

be-tween the mRNA and tRNA

Aminoacyl-tRNA Synthetases Interpret the Second Genetic Code

A second genetic code must exist, the code by which each aminoacyl-tRNA

syn-thetase matches up its amino acid with tRNAs that can interact with codons

speci-R +

A C

3 

70

5

Acceptor stem 75

65

R C



T 60

Variable loop

Anticodon loop 35

Anticodon

25 G

G



19 18

TC loop

D-loop

10

C P

A R



U Y

45 40

H 30 A

C G

Y +

FIGURE 30.1 Generalized secondary structure of tRNA molecules Circles represent nucleotides in the tRNA sequence The numbers given indicate the standardized numbering system for tRNAs (which differ in total num-ber of nucleotides) Dots indicate places where the number of nucleotides may vary in different tRNA species All tRNAs have the invariant 3-base sequence CCA at their 3 -ends Recall from Chapter 10 that tRNA molecules often have modified or unusual bases.

Overlapping code

1 2 3 4 5

Nucleic acid

Nucleic acid

etc.

Nonoverlapping code

etc.

(a)

Punctuated code

Nucleic

Continuous code 1

Nucleic

(b)

“comma” “comma” “comma”

FIGURE 30.2 (a) An overlapping versus a nonoverlap-ping code (b) A continuous versus a punctuated code.

First Third

Third-Base Degeneracy Is Color-Coded

Third Bases Third-Base with Same Relationship Meaning Number of Codons

Third base U, C, A, G 32 (8 families) irrelevant

Purines A or G 12 (6 pairs) Pyrimidines U or C 14 (7 pairs) Three out U, C, A 3 (AUX Ile)

of four Unique G only 2 (AUG Met)

Unique A only 1 (UGA Stop) definition

*AUG signals translation initiation as well as coding for Met residues.

TABLE 30.1 The Genetic Code

A DEEPER LOOK

Natural and Unnatural Variations in the Standard Genetic Code

The genomes of some lower eukaryotes, prokaryotes, and

mito-chondria show some exceptions to the standard genetic code

(Table 30.1) in codon assignments The phenomenon is more

common in mitochondria For example, the termination codon

UGA codes for tryptophan in mitochondria from various animals,

protozoans, and fungi AUA, normally an Ile codon, codes for

me-thionine in some animal and fungal mitochondrial genomes, and

AGA (an Arg codon) is a termination codon in vertebrate

mito-chondria but is a Ser codon in fruit fly mitomito-chondria

Mitochon-dria in several species of yeast use the CUX codons to specify Thr

instead of Leu Some yeast and algal mitochondria use CGG,

normally an Arg codon, as a stop codon

Less common are genomic codon variations within the

genomes of prokaryotic and eukaryotic cells Among the lower

eukaryotes, certain ciliated protozoans (Tetrahymena and

Parame-cium) use UAA and UGA as glutamine codons rather than stop

codons Instances in prokaryotes include use of the stop codon

UGA to specify Trp by Mycoplasma Perhaps most interesting is the

use of some UGA codons by both prokaryotes and eukaryotes

(in-cluding humans) to specify selenocysteine (Sec), an analog of

cys-teine in which the sulfur atom is replaced by a selenium atom

In-deed, the identification of Sec residues in proteins from bacteria,

archaea, and eukaryotes has led some people to nominate Sec as

the 21st amino acid! Sec formation requires a novel Sec-specific

tRNA known as tRNASec This tRNASecis loaded with a Ser residue

by seryl-tRNA synthetase, the aminoacyl-tRNA synthetase for

ser-ine Then, in an ATP-dependent process, the Ser-O is replaced by

Se Translation of UGA codons by selenocysteinyl-tRNASec de-pends on the presence of specific stem-loop secondary structures

in the mRNA called SECIS elements that recode the UGA codon

from “stop” to “Sec.” SECIS elements are recognized by specific proteins that recruit selenocysteinyl-tRNASecto the UGA codon during protein synthesis Most selenoproteins (proteins contain-ing selenocysteine) are involved in oxidation–reduction reac-tions, and Sec participates directly in the catalytic mechanism Sec provides a more reactive functionality than Cys

Recently, in vitro methods have been developed to introduce genetically encoded unnatural amino acids with different physi-cal, chemiphysi-cal, or biological properties into bacterial or mam-malian cells More than 30 such unnatural amino acids, each with unique chemical and photochemical reactivity, can be incorpo-rated into proteins using a unique codon and corresponding

tRNA–aminoacyl-tRNA synthetase pair (See Wang L., Xie J., and

Schultz P G., 2006 Expanding the genetic code Annual Review of

Bio-physics and Biomolecular Structure 35:225–249.)

Selenocysteine (Sec)

H

NH3+

fying its amino acid To interpret this second genetic code, an aminoacyl-tRNA

syn-thetase must discriminate between the 20 amino acids and the many tRNAs and

uniquely picks out its proper substrates—one specific amino acid and the tRNA(s)

appropriate to it—from among the more than 400 possible combinations The

ap-propriate tRNAs are those having anticodons that can base-pair with the codons

specifying the particular amino acid It is imperative that the proper amino acids be

loaded onto the various tRNAs so that the mRNA can be translated with fidelity

Al-though the primary genetic code is key to understanding the central dogma of

mo-lecular biology on how DNA encodes proteins, the second genetic code is just as

crucial to the fidelity of information transfer

Cells have 20 different aminoacyl-tRNA synthetases, one for each amino acid

Each of these enzymes catalyzes ATP-dependent attachment of its specific amino

acid to the 3-end of its cognate tRNA molecules (Figure 30.3) The products of the

reaction are an aminoacytl-tRNA, AMP, and PPi Ever-present pyrophosphatases

quickly hydrolyze the pyrophosphate product to give 2 Pi This highly exergonic

re-action provides the overall thermodynamic driving force for aminoacyl-tRNA

syn-thesis The aminoacyl-tRNA synthetase reaction serves two purposes:

1 It activates the amino acid so that it will readily react to form a peptide bond

2 It bridges the information gap between amino acids and codons

The underlying mechanisms of molecular recognition used by each

aminoacyl-tRNA synthetase to bring the proper amino acid to its cognate aminoacyl-tRNA are the

em-bodiment of the second genetic code

Evolution Has Provided Two Distinct Classes

of Aminoacyl-tRNA Synthetases

Despite their common enzymatic function, aminoacyl-tRNA synthetases are a

di-verse group of proteins in terms of size, amino acid sequence, and oligomeric

structure In higher eukaryotes, some aminoacyl-tRNA synthetases are assembled

into large multiprotein complexes The aminoacyl-tRNA synthetases fall into two

fundamental classes on the basis of similar amino acid sequence motifs, oligomeric

state, and acylation function (Table 30.2): class I and class II Class I

aminoacyl-tRNA synthetases first add the amino acid to the 2-OH of the terminal adenylate

residue of tRNA before shifting it to the 3-OH; class II enzymes add it directly to

the 3-OH (Figure 30.3) The catalytic domains of these enzymes evolved from two

different ancestral predecessors Aminoacyl-tRNA synthetases are ranked among

the oldest proteins because the different classes of these enzymes were present very

early in evolution Class I and class II aminoacyl-tRNA synthetases interact with the

tRNA 3-terminal CCA and acceptor stem in a mirror-symmetric fashion with

re-spect to each other (Figure 30.4) Class I enzymes bind to the tRNA acceptor stem

helix from the minor-groove side, whereas class II enzymes bind it from the

major-groove side

Both class I and class II aminoacyl-tRNA synthetases can be approximated as

two-domain structures, as can their L-shaped tRNA substrates, which have the

acceptor stem/CCA-3-OH at one end and the anticodon stem-loop at the other

(see Figures 11.35 and 30.5) This L-shaped tertiary structure of tRNAs separates

the 3-CCA acceptor end from the anticodon loop by a distance of 7.6 nm The

two domains of tRNAs have distinct functions: The 3-CCA end is the site of

aminoacylation, and the anticodon-containing domain interacts with the mRNA

template The two domains of tRNAs interact with the separate domains in the

synthetases One of the two major aminoacyl-tRNA synthetase domains is the

cat-alytic domain (which defines the difference between class I and class II enzymes);

this domain interacts with the tRNA 3-CCA end The other major domain in

aminoacyl-tRNA synthetases is highly variable and interacts with parts of the

tRNA beyond the acceptor-TC stem-loop domain, including, in some cases, the

anticodon

Cognate kindred; in this sense, cognate refers to

those tRNAs having anticodons that can read one

or more of the codons that specify one particular amino acid

Class I Class II

TABLE 30.2 The Two Classes of Aminoacyl-tRNA

Synthetases

Aminoacyl-tRNA Synthetases Can Discriminate Between the Various tRNAs

Aside from the need to uniquely recognize their cognate amino acids, aminoacyl-tRNA synthetases must be able to discriminate between the various aminoacyl-tRNAs The structural features that permit the synthetases to recognize and aminoacylate their

(ii)

(i)

:

(a)

E

E

P P

R CH C

+NH3

O

O–

+ tRNAR

+

Mg 2 +

H +NH3

tRNAR O

Aminoacyl-tRNA

(b)

R CH C +NH3

O

O–

+

H +NH3

O

O

Adenine

Enzyme-bound aminoacyl-adenylate

H +NH3

O

O

Adenine R

O–

Adenine

OH HO

2' 3'

H +NH3

AMP

AMP

AMP

O

O

Adenine R

+

O–

Adenine

OH

2' 3'

H +NH3

O

O

Adenine R

+

O–

Adenine

O

H +NH3 O

2 ⴕ-O aminoacyl-tRNA

O–

Adenine

OH O

3 ⴕ-O aminoacyl-tRNA

H +NH3

OH HO

HO

OH HO

OH

Transesterification

ATP

ATP

Aminoacyl-tRNA synthetase

Class I aminoacyl-tRNA synthetases

Class II aminoacyl-tRNA synthetases

FIGURE 30.3 The aminoacyl-tRNA synthetase reaction (a) The overall reaction (b) Aminoacyl-tRNA formation

pro-ceeds in two steps: (i) formation of an aminoacyl-adenylate and (ii) transfer of the activated amino acid moiety of the mixed anhydride to either the 2 -OH (class I aminoacyl-tRNA synthetases) or 3-OH (class II aminoacyl-tRNA synthetases) of the ribose on the terminal adenylic acid at the 3 -CCA terminus common to all tRNAs.Those aminoacyl-tRNAs formed as 2 -aminoacyl esters undergo a transesterification that moves the aminoacyl function

to the 3 -O of tRNA.Only the 3-esters are substrates for protein synthesis.

cognate tRNA(s) are not universal That is, a common set of rules does not govern

tRNA recognition by these enzymes Most surprising is the fact that the recognition

features are not limited to the anticodon and, in some instances, do not even

in-clude the anticodon For most tRNAs, a set of sequence elements is recognized by

its specific aminoacyl-tRNA synthetase, rather than a single distinctive nucleotide or

base pair These elements include one or more of the following: (1) at least one

base in the anticodon; (2) one or more of the three base pairs in the acceptor stem;

and (3) the base at canonical position 73 (the unpaired base preceding the CCA

end), referred to as the discriminator base because this base is invariant in the

tRNAs for a particular amino acid Figure 30.5 presents a ribbon diagram of a tRNA

FIGURE 30.4 Mirror-symmetric interactions of class I versus class II aminoacyl-tRNA synthetases with their tRNA substrates The two different classes of aminoacyl-tRNA synthetases bind to opposite faces of aminoacyl-tRNA molecules On the left is the structure of the class I glutaminyl-tRNA Gln synthetase ⬊tRNA Gln complex with a bound active-site inhibitor (orange) (pdb id  1EUQ).

On the right is the structure of the class II threonyl-tRNA Thr synthetase ⬊tRNA Thr complex with AMP (red) at the active site (pdb id  1QF6).The relative orientation

of the tRNA is the same in both structures, with the

3 -CCA end of the tRNA pointed away from the viewer.

TC stem loop

Variable

pocket

Variable

loop

A, F, L, R

F

60

20

42

10

32

50

70

1

Acceptor stem

A, D, G, H, M

Q, S, T, V, W

Position 73

A, C, D, E, F, G

H, I, K, L, M, N, P

Q, R, S, V, W, Y

D stem loop

Anticodon stem loop

Anticodon

C, D, E, F, G

H, I, K, M, N, P

Q, R, T, V, W, Y

Variable stem loop

S, Y

FIGURE 30.5 Ribbon diagram of tRNA tertiary structure Numbers represent the consensus nucleotide sequence (see Figure 30.1) The locations of nucleotides recog-nized by the various aminoacyl-tRNA synthetases are indicated; shown within the boxes are one-letter desig-nations of the amino acids whose respective aminoacyl-tRNA synthetases interact at the discriminator base (po-sition 73), acceptor stem, variable pocket and/or loop, or anticodon The inset shows additional recognition sites

in those tRNAs having a variable loop that forms a stem-loop structure (Adapted from Figure 2 in Saks, M E., Sampson,

J R., and Abelson, J N., 1994 The transfer RNA problem: A search

for rules Science 263:191–197.)

molecule showing the location of nucleotides that contribute to specific recogni-tion by the respective aminoacyl-tRNA synthetases for each of the 20 amino acids Interestingly, the same set of tRNA features that serves as positive determinants for binding and aminoacylation of the tRNA by its cognate aminoacyl-tRNA synthetase may act as negative determinants that prohibit binding and aminoacylation by other (noncognate) aminoacyl-tRNA synthetases Because no common set of rules exists,

the second genetic code is an operational code based on aminoacyl-tRNA

synthe-tase recognition of varying sequence and structural features in the different tRNA molecules during the operation of aminoacyl-tRNA synthesis Some examples of this code are given in Figure 30.6

Escherichia coli Glutaminyl-tRNAGlnSynthetase Recognizes Specific Sites on tRNAGln

E coli glutaminyl-tRNAGlnsynthetase, a class I enzyme, provides a good illustration of aminoacyl-tRNA synthetase⬊cognate tRNA interactions This glutaminyl-tRNAGln syn-thetase shares a continuous interaction with its cognate tRNA that extends from the anticodon to the acceptor stem along the entire inside of the L-shaped tRNA (Fig-ure 30.7) Specific recognition elements include enzyme contacts with the discrimi-nator base, acceptor stem, and anticodon, particularly the central U in the CUG an-ticodon The carboxylate group of Asp235makes sequence-specific H bonds in the tRNA minor groove with the 2-NH2group of G3 in the base pair G3⬊C70 of the ac-ceptor stem A mutant glutaminyl-tRNAGlnsynthetase with Asn substituted for Asp at position 235 shows relaxed specificity; that is, it now will acylate noncognate tRNAs with Gln

The Identity Elements Recognized by Some Aminoacyl-tRNA Synthetases Reside in the Anticodon

Alteration of the anticodons of either tRNATrp or tRNAValto CAU, the anticodon for the methionine codon AUG, transforms each of the tRNAs into a substrate for methionyl-tRNA synthetase, and they are loaded with methionine Similarly, re-versing the methionine CAU anticodon of tRNAMetto UAC transforms it into a sub-strate for valyl-tRNAVal synthetase Clearly, methionyl-tRNA synthetase and valyl-tRNA synthetase rely on the anticodon in selecting valyl-tRNAs for loading

A Single G ⬊U Base Pair Defines tRNAAlas

The noncanonical base pair, G3⬊U70, is the singular feature by which alanyl-tRNAAlasynthetase recognizes tRNAs as its substrates All tRNAAlarepresentatives, from archaea to eukaryotes, possess this G3⬊U70 acceptor stem base pair Alter-ing this unusual G3⬊U70 base pair of tRNAAlato G⬊C, A⬊U, or even U⬊G abol-ishes its ability to be aminoacylated with alanine On the other hand, provided the G3⬊U70 base pair is present, alanyl-tRNAAla synthetase aminoacylates a 24-nucleotide stem-loop analog of tRNAAla (Figure 30.8) The key feature of the G3⬊U70 base pair is the 2-NH2 group of G3 In the RNA A-form double-helical structure adopted by the tRNA acceptor stem, the G3 2-HN2group is exposed in the minor groove of the helix, and if the G3 pairing partner is a U, this 2-NH2

group lacks an H-bonding partner (Figure 30.8) Thus, an unpaired G 2-amino group at the right place in a tRNA acceptor stem marks a tRNA for aminoacyla-tion by alanyl-tRNAAlasynthetase

30.3 What Are the Rules in Codon–Anticodon Pairing?

Protein synthesis depends on the codon-directed binding of the proper aminoacyl-tRNAs so that the right amino acids are sequentially aligned according to the spec-ifications of the mRNA undergoing translation This alignment is achieved via

20

tRNA Phe (yeast) tRNA Met

f

11

70 3

24

FIGURE 30.6 Major identity elements in four tRNA

species Each base in the tRNA is represented by a circle.

Numbered filled circles indicate positions of identity

elements within the tRNA that are recognized by its

specific aminoacyl-tRNA synthetase (Adapted from

Schul-man, L H., and Abelson, J., 1988 Recent excitement in

under-standing transfer RNA identity Science 240:1591–1592.)

FIGURE 30.7 Structure of E coli glutaminyl-tRNAGln

synthetase complexed with tRNA Gln and ATP (pdb id 

1GSG) The protein ⬊tRNA contact region extends along

one side of the entire length of this extended protein.

The acceptor stem of the tRNA and the ATP (green) fit

into a cleft at the top of the protein in this view The

enzyme also interacts extensively with the anticodon

(lower tip of tRNA Gln ).

codon–anticodon pairing in antiparallel orientation (Figure 30.9) However,

con-siderable degeneracy exists in the genetic code at the third position Conceivably,

this degeneracy could be handled in either of two ways: (1) Codon–anticodon

recognition could be highly specific so that a complementary anticodon is

re-quired for each codon, or (2) fewer than 61 anticodons could be used for the

“sense” codons if certain allowances were made in the base-pairing rules Then,

some anticodons could recognize more than one codon As early as 1965, it was

known that poly(U) bound all Phe-tRNAPhemolecules even though UUC is also a

Phe codon The phenylalanine-specific tRNAs could recognize either UUU or

UUC Also, one particular yeast tRNAAlawas able to bind to three codons: GCU,

GCC, and GCA

Francis Crick Proposed the “Wobble” Hypothesis

for Codon–Anticodon Pairing

Francis Crick considered these results and tested alternative base-pairing

possibili-ties by model building He hypothesized that the first two bases of the codon and

the last two bases of the anticodon form canonical Watson–Crick A⬊U or G⬊C base

pairs, but pairing between the third base of the codon and the first base of the

an-ticodon follows less stringent rules That is, a certain amount of play, or wobble,

might be allowed in base pairing at this position The third base of the codon is

sometimes referred to as the wobble position.

Crick’s investigations suggested a set of rules for pairing between the third base

of the codon and the first base of the anticodon (Table 30.3) The wobble rules

in-dicate that a first-base anticodon U could recognize either an A or G in the codon

third-base position; first-base anticodon G might recognize either U or C in the

third-base position of the codon; and first-base anticodon I1might interact with U,

C, or A in the codon third position (Figure 30.10).2

The wobble rules also predict that four-codon families (like Pro or Thr), where

any of the four bases may be in the third position, require at least two different

tRNAs However, all members of the set of tRNAs specific for a particular amino

acid—termed isoacceptor tRNAs—are served by one aminoacyl-tRNA synthetase.

20

15

40

60 70

70 76

30

G A

A

A

U

A

C

U C

C C C C

G U

G G G C A

G

G

A

U

A A A

U U U C

C C C C

G

C C

G G

C

G

G U U

A U

G G

G G

G C C G C

C U

A A A

C U A G C ψ T

C

G

G

D

A

A

A G

C GA

G

U

C

C

C G

G tRNA Ala/GGC

(a)

Microhelix Ala

U

1

10

N

O

R

N

H H

N O

N R O

N H

H

Major gr

(b)

oove

Minor groove

G–U

FIGURE 30.8 (a) A microhelix analog of tRNAAla is aminoacylated by alanyl-tRNA Ala synthetase, provided it has the characteristic tRNA Ala

G3 ⬊U70 acceptor stem base pair.The microhelix Ala consists of nucleo-tides 1 through 13 of tRNA Ala/GGC connected directly to 66 through 76 to re-create the tRNA Ala7-bp acceptor stem (b) The unbonded 2-HN2

group of the G3 in the G3 ⬊U70 base pair would lie within the minor groove of an A-form RNA double helix [(a) Adapted from Schimmel, P., 1989.

Parameters for molecular recognition of transfer RNAs Biochemistry 28:2747–2759.]

3 

5 

5 

3  Anticodon tRNA

mRNA Codon

FIGURE 30.9 Codon–anticodon pairing Complementary trinucleotide sequence elements align in antiparallel fashion.

1 I is inosine (6-OH purine).

2 Thus, the first base of the anticodon indicates whether the tRNA can read one, two, or three

differ-ent codons: Anticodons beginning with A or C read only one codon, those beginning with G or U read

two, and anticodons beginning with I can read three codons.

Bases Recognized Base on the Anticodon on the Codon

Adapted from Crick, F H C., 1966 Codon–anticodon pairing:

The wobble hypothesis Journal of Molecular Biology

19:548–555.

TABLE 30.3 Base-Pairing Possibilities at the

Third Position of the Codon

Some Codons Are Used More Than Others

Because more than one codon exists for most amino acids, the possibility for varia-tion in codon usage arises Indeed, variavaria-tion in codon usage accommodates the fact that the DNA of different organisms varies in relative A⬊T/G⬊C content Neverthe-less, even in organisms of average base composition, codon usage may be biased

Table 30.4 gives some examples from E coli and humans reflecting the nonrandom

usage of codons Of more than 109,000 Leu codons tabulated in a set of human genes, CUG was used in excess of 48,000 times, CUC more than 23,000 times, but UUA just 6000 times

The occurrence of codons in E coli mRNAs correlates well with the relative

abun-dance of the tRNAs that read them Preferred codons are represented by the most abundant isoacceptor tRNAs Furthermore, mRNAs for proteins that are synthe-sized in abundance tend to employ preferred codons Rare tRNAs correspond to rarely used codons, and messages containing such codons might experience delays

in translation

Nonsense Suppression Occurs When Suppressor tRNAs Read Nonsense Codons

Mutations that alter a sense codon to one of the three nonsense codons—UAA, UAG,

or UGA—result in premature termination of protein synthesis and the release of trun-cated (incomplete) polypeptides Geneticists found that second mutations elsewhere

in the genome were able to suppress the effects of nonsense mutations so that the

or-ganism survived, a phenomenon termed nonsense suppression The molecular basis for nonsense suppression was a mystery until it was realized that suppressors were

mu-tations in tRNA genes that altered the anticodon so that the mutant tRNA could now read a particular “stop” codon and insert an amino acid For example, alteration of the anticodon of a tRNATyrfrom GUA to CUA allows this tRNA to read the amber stop codon, UAG, and insert Tyr (The nonsense codons are named amber [UAG], ochre

[UAA], and opal [UGA]) Suppressor tRNAs are typically generated from minor tRNA

species within a set of isoacceptor tRNAs, so their recruitment to a new role via muta-tion does not involve loss of an essential tRNA; that is, the mutamuta-tion is not particularly

N N N

N N N

N

N

H

H Uracil

H

H

Ribose

Ribose

Ribose

O

O

N

N

N N

H H

Ribose O

N N N

N N

N

H

H

H

H

Ribose

O

O O

FIGURE 30.10 Pairing of anticodon inosine (I, left) with C,

U, or A as the codon third base Note that I is in the keto

tautomeric form.

deleterious to the organism A suppressor tRNA, as a mutant tRNA, may even carry and

introduce an amino acid different from the one borne by the wild-type tRNA

30.4 What Is the Structure of Ribosomes,

and How Are They Assembled?

Protein biosynthesis is achieved by the process of translation Translation converts

the language of genetic information embodied in the base sequence of a

messen-ger RNA molecule into the amino acid sequence of a polypeptide chain During

translation, proteins are synthesized on ribosomes by linking amino acids together

in the specific linear order stipulated by the sequence of codons in an mRNA

Ri-bosomes are the agents of protein synthesis

cells, as well as in the matrix of mitochondria and the stroma of chloroplasts The

gen-eral structure of ribosomes is described in Chapter 10; here we consider their

struc-ture in light of their function in synthesizing proteins Ribosomes are

mechanochem-ical systems that move along mRNA templates, orchestrating the interactions between

successive codons and the corresponding anticodons presented by aminoacyl-tRNAs

As they align successive amino acids via codon–anticodon recognition, ribosomes also

catalyze the formation of peptide bonds between the growing peptide chain and

in-coming amino acids

Prokaryotic Ribosomes Are Composed of 30S and 50S Subunits

E coli ribosomes are representative of the structural organization of the

prokary-otic versions of these supramolecular protein-synthesizing machines (Table 30.5,

see also Figure 10.22) The E coli ribosome is a roughly globular particle with a

The results are expressed as frequency of occurrence of a codon per 1000 codons

tabulated in 1562 E coli genes and 2681 human genes, respectively (Because E coli

and human proteins differ somewhat in amino acid composition, the frequencies for

a particular amino acid do not correspond exactly between the two species.)

Adapted from Wada, K., et al., 1992 Codon usage tabulated from Genbank genetic sequence data Nucleic Acids Research

20:2111–2118.

TABLE 30.4 Representative Examples of Codon Usage in E.coli and Human Genes

diameter of 25 nm, a sedimentation coefficient of 70S, and a mass of about

2520 kD It consists of two unequal subunits that dissociate from each other at Mg2

concentrations below 1 mM The smaller, or 30S, subunit is composed of 21

differ-ent proteins and a single rRNA, 16S ribosomal RNA (rRNA) The larger 50S sub-unit consists of 31 different proteins and two rRNAs: 23S rRNA and 5S rRNA

Ri-bosomes are roughly two-thirds RNA and one-third protein by mass An E coli cell

contains around 20,000 ribosomes, constituting about 20% of the dry cell mass

Prokaryotic Ribosomes Are Made from 50 Different Proteins and Three Different RNAs

Ribosomal Proteins There is one copy of each ribosomal protein per 70S ribosome, excepting protein L7/L12 (L7 and L12 have identical amino acid se-quences and differ only in the degree of N-terminal acetylation) Only one pro-tein is common to both the small and large subunit: S20 L26 The largest ribosomal protein is S1 (557 residues, 61.2 kD); the smallest is L34 (46 residues, 5.4 kD) The sequences of ribosomal proteins share little similarity These pro-teins are typically rich in the cationic amino acids Lys and Arg and have few aro-matic amino acid residues, properties appropriate to proteins intended to inter-act strongly with polyanionic RNAs

Ribosome Small Subunit Large Subunit

Protein number 21 polypeptides* 31 polypeptides†

TABLE 30.5 Structural Organization of E.coli Ribosomes

Operon

rrnA

rrnB

rrnC

rrnD

rrnE

rrnG

rrnH

Spacer tRNA Ile, Ala Glu Glu Ile, Ala Glu Glu Ile, Ala

Trailer tRNA

-Asp, Trp

-Asp

Chromosomal location (min) 86 89 84 71 90 56 5

RNase III RNase III

RNase III RNase III

5S

23S 16S

FIGURE 30.11 The seven ribosomal RNA operons in

E coli Numerals to the right of the brackets indicate the

number of species of tRNA encoded by each transcript.

*The S proteins

† The L proteins