Hogg S. Essential microbiology

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Microbial Genetics

A gene is a sequence

of DNA that usually en-

codes a polypeptide.

When any living organism reproduces, it passes on

genetic information to its offspring. This information

takes the form of genes, linear sequences of DNA that

can be thought of as the basic units of heredity. The total

complement of an organism’s genetic material is called

its genome.

How do we know genes

are made of DNA?

The concept of the gene as an inherited physical entity determining some aspect of

an organism’s phenotype dates back to the earliest days of genetics. The question of

what genes are actually made of was a major concern of molecular biologists (not that

they would have described themselves as such!) in the ﬁrst half of the 20th century.

Since it was recognised by this time that genes must be located on chromosomes, and

that chromosomes (in eucaryotes) comprised largely protein and DNA, the reasonable

assumption was made that genes must be made up of one of these substances. In the

early years, protein was regarded as the more likely candidate, since, from what was

known of molecular structure at the time, it offered far more scope for the variation

which would be essential to account for the thousands of genes that any organism must

possess. The road to proving that DNA is in fact the ‘stuff of life’ was a long and hard

one, which can be read about elsewhere; we shall mention below just some of the key

experiments which provided crucial evidence.

In 1928 the Englishman Fred Grifﬁth carried out a seminal series of experiments

which not only demonstrated for the ﬁrst time the phenomenon of genetic transfer

in bacteria (a subject we shall consider in more detail later in this chapter), but also

acted as the ﬁrst step towards proving that DNA was the genetic material. As we shall

see, Grifﬁth showed that it was possible for heritable characteristics to be transferred

from one type of bacterium to another, but the cellular component responsible for this

phenomenon was not known at this time.

Attempts were made throughout the 1930s to isolate and identify the transform-

ing principle, as it became known, and in 1944 Avery, MacLeod and McCarty pub-

lished a paper, which for the ﬁrst time, proposed DNA as the genetic material. Avery

and his colleagues demonstrated that when DNA was rendered inactive by enzy-

matic treatment, transforming ability was lost from a cell extract, but if proteins,

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270 MICROBIAL GENETICS

Box 11.1 Hershey and Chase: the Waring blender experiment

In 1952 Alfred Hershey and Martha Chase provided further experimental evidence

that DNA was the genetic material. In their experiment, the bacteriophage T2 was

grown with E. coli cells in a medium with radiolabelled ingredients, so that their pro-

teins contained

S, and their DNA

P. The phages were harvested, and mixed with

a fresh culture of E. coli. They were left long enough for the phage particles to infect

the bacteria, (but not long enough to produce new phage particles and lyse the

cells). The culture was then subjected to mechanical agitation in a Waring blender,

which, it was hoped, would remove the ‘shell’of the phages from the outside of the

bacteria, but leave the injected genetic material inside.

T2 phage

DNA

labelled

Protein

labelled

Infect bacteria in

labelled media

Isolate phage

Infect bacteria in

unlabelled media

Separate phage ghosts

from bacterial cells

Ghost

Ghosts

unlabelled

Bacteria labelled

with

Ghosts labelled

with

Bacteria

unlabelled

From Reece, RJ: Analysis of Genes and Genomes, John Wiley & Sons, 2003. Repro-

duced by permission of the publishers

The bacteria were sedimented by centrifugation, leaving the much lighter phage

‘shells’ in the supernatant. When solid and liquid phases were analysed for

P and

S, it was found that nearly all the

P was associated with the bacterial cells, while

the great majority of the

S remained in the supernatant. The conclusion drawn

from these results is that it was the

P-labelled DNA which had been injected into

the bacteria, and which was therefore the genetic material.

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DNA REPLICATION 271

Figure 11.1 DNA replication is semiconservative. Following replication, each new DNA

molecule comprises one strand from the parent DNA and one newly synthesised strand

carbohydrates or any other cellular component was similarly inactivated, the ability

was retained. In spite of this apparently convincing proof, the pro-protein lobby was

not easily persuaded. It was to be several more years before the experimental results of

Alfred Hershey and Martha Chase (Box 11.1) coupled with Watson and Crick’s model

for DNA structure (Figure 2.23) ﬁnally cemented the universal acceptance of DNA’s

central role in genetics.

DNA replication

The elucidation of the structure of DNA by Watson and Crick in 1953 stands as one

of the great scientiﬁc breakthroughs of the 20th century. An important implication of

their model lay in the complementary nature of the two strands, as they commented

themselves in their famous paper in Nature:

It has not escaped our notice that the speciﬁc pairing we have postulated immediately suggests a

possible copying mechanism for the genetic material.

The mechanism they proposed became known as the semiconservative replication of

DNA, so called because each daughter molecule comprises one parental strand and one

newly synthesised strand (Figure 11.1). The parental double helix of DNA unwinds and

each strand acts as a template for the production of a new complementary strand, with

new nucleotides being added according to the rules of base-pairing. In 1957, 4 years

after the publication of Watson and Crick’s model, Matthew Meselson and Franklin

Stahl provided experimental proof of the semiconservative nature of DNA replication

(Box 11.2). The exact way in which replication takes place in procaryotic and eucaryotic

cells differs in some respects, but we shall take as our model replication in E. coli, since

it has been studied so extensively.

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Box 11.2 Experimental proof of semiconservative replication

The semiconservative model of DNA replication, as suggested by Watson and Crick,

was not the only one in circulation during the 1950s. Meselson and Stahl provided

experimental evidence which not only supported the semiconservative model, but

also showed the other models to be unworkable. Their elegantly designed exper-

iment used newly developed techniques to differentiate between parental DNA

and newly synthesised material.

First, they grew E. coli in a medium with ammonium salts containing the heavy

isotope

N as the only source of nitrogen. This was done for several generations of

growth, so that all the cells contained nitrogen exclusively in the heavy form.

The bacteria were then transferred to a medium containing nitrogen in the nor-

mal,

N form. After a single round of replication, DNA was isolated from the culture

and subjected to density gradient centrifugation. This is able to differentiate be-

tween DNA containing the two forms of nitrogen, as

N has a greater buoyant

density and therefore settles at a lower position in the tube.

Meselson and Stahl found just a single band of DNA after centrifugation, with a

density intermediate between that of

N and

N, indicating a hybrid molecule, as

predicted by the semiconservative model. After a second round of E. coli replication

in a medium containing

N, two bands of DNA were produced, one hybrid and one

containing exclusively

N, exactly as predicted by the semiconservative model, but

inconsistent with other hypotheses.

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DNA REPLICATION 273

DNA replication in procaryotes

You may recall from Chapter 4 that bacteria multiply by a process of binary ﬁssion;

before this occurs, each cell must duplicate its genetic information so that each daughter

cell has a copy.

DNA replication involves the action of a number of specialised enzymes:

helicases

DNA topoisomerases

DNA polymerase I

DNA polymerase III

DNA primase

DNA ligase.

DNA replication takes

place at a replication

fork, a Y-shaped struc-

ture formed by the sep-

arating strands. The fork

moves along the DNA as

replication proceeds.

Replication begins at a speciﬁc sequence called an ori-

gin of replication. The two strands of DNA are caused to

separate by helicases (Figure 11.2), while single-stranded

DNA binding proteins (SSB) prevents them rejoining.

Opening out part of the double helix causes increased

tension (supercoiling) elsewhere in the molecule, which

is relieved by the enzyme DNA topoiosomerase (some-

times known as DNA gyrase). As the ‘zipper’ moves

along, and more single stranded DNA is exposed, DNA

polymerase III adds new nucleotides to form a comple-

mentary second strand, according to the rules of base-

pairing. DNA polymerases are not capable of initiating

the synthesis of an entirely new strand, but can only

extend an existing one. This is because they require a

free 3



-OH group onto which to attach new nucleotides.

Thus, DNA polymerase III can only work in the 5



–3



direction. A form of RNA polymerase called DNA pri-

mase synthesises a short single strand of RNA, which

can be used as a primer by the DNA polymerase III.

(Figure 11.2).

A primer is a short

sequence of single-

stranded DNA or RNA

required by DNA poly-

merase as a starting

point for chain exten-

sion.

When replication occurs, complementary nucleotides are added to one of the strands

(the leading strand) in a continuous fashion (Figure 11.2). The other strand (the lagging

strand), however, runs in the opposite polarity; so how is a complementary sequence

synthesised here? The answer is that DNA polymerase III allows a little unwinding to

take place and then, starting at the fork, works back over from a new primer, in the 5



–3



direction. Thus, the second strand is synthesised discontinuously, in short bursts, about

1000–2000 nucleotides at a time. These short stretches of DNA are called Okazaki

fragments, after their discoverers.

On the lagging strand, a new RNA primer is needed at the start of every Okazaki

fragment. These short sequences of RNA are later removed by DNA polymerase I, which

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274 MICROBIAL GENETICS

single-stranded

binding protein

5′

3′

5′

3′

5′

3′

3′ 5′

helicase

RNA primer

leading strand

DNA polymerase III

Okazaki

fragment

lagging strand

DNA

primase

DNA

polymerase

Figure 11.2 DNA replication takes place at a replication fork. Strands of DNA are sep-

arated and unwound by helicase and DNA gyrase, and prevented from rejoining by the

attachment of single-strand binding proteins. Starting from an RNA primer, DNA poly-

merase III adds the complementary nucleotides to form a second strand. On the leading

strand, a single primer is set down (not shown), and replication proceeds uninterrupted in

the same direction as the fork. Because DNA polymerase III only works in the 5



→ 3



di-

rection, on the lagging strand a new primer must be added periodically as the strands open

up. Replication here is thus discontinuous, as a series of Okazaki fragments (see the text).

From Bolsover, SR, Hyams, JS, Jones, S, Shepherd, EA & White, HA: From Genes to Cells,

John Wiley & Sons, 1997. Reproduced by permission of the publishers

DNA ligase repairs

breaks in DNA by re-

establishing phosphodi-

ester bonds in the sugar-

phosphate backbone.

then replaces them with DNA nucleotides. Finally, the

fragments are joined together by the action of DNA

ligase. Replication is bidirectional (Figure 11.3), with

two forks moving in opposite directions; when they

meet, the whole chromosome is copied and replication

is complete

What happens when replication goes wrong?

It is clearly important that synthesis of a complementary second strand of DNA should

occur with complete accuracy, but occasionally, a non-complementary nucleotide is

inserted; this may happen as frequently as once in every 10 000 nucleotides. Cells,

however, are able to have a second attempt to incorporate the correct base because of

the proofreading activity of the enzymes DNA polymerase I and III. These are able to

This rather long-winded description hardly does justice to a process that incorporates new nucleotides at a

rate of around 1000 per second!

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WHAT EXACTLY DO GENES DO? 275

Direction of replication fork

Replication bubble

Figure 11.3 DNA replication is bidirectional. Two replication forks form simultaneously,

moving away from each other and developing a replication bubble

cut out the ‘wrong’ nucleotide and replace it with the correct one. As a result of this

monitoring, mistakes are very rare; they are thought to occur at a frequency of around

one in every billion (10

) nucleotides copied. Mistakes that do slip through the net result

in mutations, which are discussed later in this chapter.

DNA replication in eucaryotes

In eucaryotic organisms, the DNA is linear, not circular, so the process of replication

differs in some respects. Genome sizes are generally much bigger in eucaryotes, and

replication much slower, so numerous replication forks are active simultaneously on

each chromosome. Replication takes place bidirectionally, creating numerous replica-

tion bubbles (Figure 11.4) which merge with one another until the whole chromosome

has been covered.

What exactly do genes do?

At the start of the 20th century Archibald Garrod had proposed that inherited disorders

such as alkaptonuria may be due to a defect in certain key metabolic enzymes, thus

offering for the ﬁrst time an explanation of how genetic information is expressed. His

ideas were not really developed however, until the work of George Beadle and Edward

Tatum in the 1940s, whose experiments with the bread mould Neurospora led to the

formulation of the one gene, one enzyme hypothesis. Although now acknowledged to be

Figure 11.4 DNA replication in eucaryotes. Many replication bubbles develop simultane-

ously; they extend towards each other and eventually merge. The arrows denote the direction

of the replication forks

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276 MICROBIAL GENETICS

Box 11.3 One gene, one enzyme: not quite true

Beadle and Tatum proposed that each gene was responsible for the production of

a specific enzyme. However all proteins, not just enzymes, are encoded by DNA,

and furthermore, some have a quaternary structure (see Chapter 2) with different

polypeptide subunits being encoded by different genes. The hypothesis was there-

fore modified to one gene, one polypeptide. Later still, it emerged that even this

is not always the case, as some genes do not encode proteins at all, but forms of

RNA.

somewhat over-generalised (see Box 11.3), this model proved useful in the years when

the molecular basis of gene action was being elucidated.

Having established that genes are made of DNA, and having a model for the structure

of DNA that explained how it was able to copy itself, the way was open in the 1950s

for scientists to work out the mechanism by which the information encoded in a DNA

sequence was converted into a speciﬁc protein.

How does a gene direct the synthesis of a protein?

You may recall from Chapter 2 that both DNA and proteins are polymers whose ‘build-

ing blocks’ (nucleotides and amino acids respectively) can be put together in an almost

inﬁnite number of sequences. The sequence of amino acids making up the primary

structure of a protein is determined by the sequence of nucleotides in the particular

gene responsible for its production. It does this not directly, but through an intermedi-

ary molecule, now known to be a form of RNA called messenger RNA (mRNA). It is this

intermediary that carries out the crucial task of passing the information encoded in the

DNA sequence to the site of protein synthesis. This unidirectional ﬂow of information

can be summarised:

DNA mRNA

DNA replication

Protein

and is often referred to as the Central Dogma of biology, because of its applicability to

all forms of life. Proposed by Crick in the late 1950s, this is still accepted as being a

true model of the basic events in protein synthesis. Sometimes the message encoded in

DNA is transcribed into either ribosomal RNA (rRNA) or transfer RNA (tRNA); these

types of RNA are not translated into proteins but represent end-products in themselves.

(In the last chapter, we saw that retroviruses have proved an exception to one part of

the Central Dogma, since they possess enzymes capable of forming DNA from an RNA

template.)

The conversion of information encoded as DNA into the synthesis of a polypeptide

chain occurs in two distinct phases (Figure 11.5); ﬁrst the ‘message’ encoded in the

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WHAT EXACTLY DO GENES DO? 277

Figure 11.5 Transcription and translation. The sequence on one strand of DNA is tran-

scribed as a molecule of mRNA (with uracil replacing thymine). The triplet code on the

mRNA is then translated at ribosomes as a series of amino acids

Messenger RNA (mRNA)

is formed from a DNA

template (transcription)

and carries its encoded

message to the ribo-

some, where it directs

synthesis of a polypep-

tide (translation).

DNA sequence of a gene is converted to mRNA by tran-

scription, then this directs the assembly of a speciﬁc se-

quence of amino acids during translation. We shall dis-

cuss how this happens shortly, but ﬁrst we need to con-

sider the question: how does the sequence of nucleotides

in a gene serve as an instruction in the synthesis of pro-

teins?

The genetic code

Messenger RNA carries information (copied from a DNA template) in the form of a

genetic code that directs the synthesis of a particular protein. The nature of this code

was worked out in the early 1960s by Marshall Nirenberg, Har Gobind Khorana and

others (Box 11.4). The message encoded in a gene takes the form of a series of triplets

(codons). Of the 64 possible three-letter combinations of A, C, G and U, 61 correspond

to speciﬁc amino acids, while the remaining three act as ‘stop’ messages, indicating that

reading of the message should cease at that point (Figure 11.6). It is essential that the

reading of the message starts at the correct place, otherwise the reading frame (groups of

Box 11.4 The genetic code is (almost) universal

The genetic code was first worked out using E. coli, but soon found to apply to other

organisms too. It seemed reasonable to assume that the code was universal, i.e.

applicable to all life forms. It has been shown, however, that certain genes such

as those found in the mitochondria of some eucaryotes employ a slight variation of

the code. Mitochondria have their own transcription enzymes, ribosomes and tRNAs

and so are able to use a modified system.