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Molecular Biology: General theory

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Molecular Biology: General Theory

Author: Dr Darshana Morar

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTS

INTRODUCTION ........................................................................................................................................... 3

NUCLEIC ACIDS .......................................................................................................................................... 3

DNA .......................................................................................................................................................... 3

RNA Structure .......................................................................................................................................... 6

GENOME ORGANIZATION ......................................................................................................................... 6

Viruses ..................................................................................................................................................... 7

Prokaryotes .............................................................................................................................................. 7

Eukaryotes ............................................................................................................................................... 8

THE CENTRAL DOGMA OF MOLECULAR BIOLOGY .............................................................................. 9

DNA REPLICATION ................................................................................................................................... 10

The replication process in bacteria ........................................................................................................ 10

The replication process in viruses .......................................................................................................... 14

The replication process in Eukaryotes ................................................................................................... 15

TRANSCRIPTION ....................................................................................................................................... 16

Prokaryotes ............................................................................................................................................ 18

Eukaryotes ............................................................................................................................................. 18

TRANSLATION .......................................................................................................................................... 19

http://creativecommons.org/licenses/by/3.0/


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REFERENCES ............................................................................................................................................ 21

The relevant websites as indicated in the course outline: ..................................................................... 21

A few general references: ...................................................................................................................... 21


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INTRODUCTION

Before the reader can gain an understanding of the practical applications of nucleic acids and proteins, the basic structure, mode of action and functions of these complex structures should be known.

Two types of nucleic acids can be found in a eukaryotic cell; deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Deoxyribonucleic acid contains the genetic instructions needed to construct components of cells, such as RNA and protein molecules. Therefore, DNA is used in the development and functioning of all known living organisms and some viruses, with the primary role being long-term storage of information. Genes are the DNA segments that carry this genetic information.

RNA differs in structure from DNA, but is transcribed (made) from DNA by enzymes called RNA polymerases and is generally processed further by other enzymes. RNA is central to the translation (synthesis) of some RNAs into proteins.

Proteins are large organic compounds made of amino acids arranged in a linear chain, joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in every process within cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism.

NUCLEIC ACIDS

DNA

DNA is made up of nucleotides. A nucleotide consists of a nitrogenous base (either a purine or a pyrimidine), a five-carbon sugar (deoxyribose), and a phosphate group. The four nitrogenous bases found in DNA are adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T).

http://en.wikipedia.org/wiki/Cell_%28biology%29


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Figure 1 Basic structure of a nucleotide

(Adapted from http://www.duke.edu/web/MAT/jennifer_sohn/unit/translation_oh.htm)

DNA is composed of a series of nucleotides, held together in a long linear chain by a phosphodiester linkage from the 3’ hydroxyl group of one sugar to the 5’ phosphate group of the next (a DNA strand or polynucleotide therefore comprises a sugar-phosphate backbone with attached bases). Since the phosphodiester bonds form between the third and fifth carbon atoms of adjacent sugars, a strand of DNA has a direction or orientation.

Most DNA is double-stranded: two chains that constitute a double helix. The two chains have opposite orientations and are connected via hydrogen bridges between opposing base groups. Each type of base on one strand forms a bond with just one type of base on the other strand. Adenine (purine) bonds only to thymine (pyrimidine) by two hydrogen bonds, and guanine (purine) bonds only to cytosine (pyrimidine) by three hydrogen bonds. This arrangement of two nucleotides binding together across the double helix is called a base pair. The sequences of the two DNA chains are thus complementary.


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Figure 2 Basic structure of DNA

By convention the upper strand is written from 5' (five prime) to 3' (three prime). If DNA is transcribed, the upper strand (known as the sense strand) corresponds to the RNA sequence. If this is messenger RNA (mRNA) it contains the triplets of the genetic code, the coding strand. Normally only this upper strand is listed in publications and databases.

In schematic drawings of gene structure the DNA is often represented as a straight line (or a circle for a plasmid). Left to right always corresponds to the 5' to 3' orientation of the upper strand.

5' _______________________ 3'

This representation does not do justice to the double-helix structure, but is nevertheless adequate to understand the DNA manipulations described in the other modules.

Cleavage of DNA always generates one end with a phosphate group coupled to the fifth carbon atom in the ribose sugar ring (the 5'-phosphate) and the other end with a hydroxyl group attached to the third carbon atom in the ribose sugar ring (the free 3'-OH group):

5'-phosphate________________________3'-OH


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RNA Structure

RNA is a very labile molecule and, like DNA, it is made from a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate. Note that the sugar present in a DNA molecule is deoxyribose, while the sugar present in a molecule of RNA is ribose (in deoxyribose there is no hydroxyl group attached to the second carbon atom in the ribose sugar ring). The extra hydroxyl group makes RNA more prone to hydrolysis which is why it is less stable than DNA. The four bases found in RNA are adenine (A), cytosine (C), guanine (G) and uracil (U) (replacing the thymine (T) in DNA).

Figure 3 Deoxyribose versus ribose sugars

Although RNA is essentially single-stranded, most biologically active RNA molecules contain self-complementary sequences that allow parts of the molecule to fold and pair with itself to form double helices. There are three major types of RNA: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). There are a number of other types of RNA present in smaller quantities as well, including small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and the 4.5S signal recognition particle (SRP) RNA. Novel species of RNA continue to be identified.

GENOME ORGANIZATION

All of the genetic information or hereditary material possessed by an organism is known as its genome. Viruses, prokaryotes and eukaryotes contain nucleic acids, with the relative amount of DNA and/or RNA differing depending on the organism. Viruses have the smallest genomes. Prokaryotes have larger genomes than viruses, while eukaryotes have the largest genomes of all.

http://en.wikipedia.org/wiki/Nucleotide

http://en.wikipedia.org/wiki/Nucleobase

http://en.wikipedia.org/wiki/Ribose

http://en.wikipedia.org/wiki/Phosphate


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Viruses

Viruses contain either DNA or RNA, never both, have no cytoplasm or cell organelles and are obligate intracellular parasites.

Viral genomes

The nucleic acid may be single-stranded or double-stranded; it may be linear, circular or segmented configuration. Single-stranded virus genomes may be:

• Positive (+) sense, i.e. of the same polarity (nucleotide sequence) as mRNA

• Negative (-) sense or

• Ambisense - a mixture of the two.

Prokaryotes

There are different groups of prokaryotes, distinguished from one another by characteristic genetic and biochemical features:

• The bacteria, which include most of the commonly encountered prokaryotes such as the Gram-negatives (e.g. Escherichia coli), the Gram-positives (e.g. Bacillus subtilis), the cyanobacteria (e.g. Anabaena) and many more.

• The archaea, which are less well-studied, and have mostly been found in extreme environments such as hot springs, brine pools and anaerobic lake bottoms.

Prokaryotic genomes

The traditional view has been that an entire prokaryotic genome is contained in a single circular DNA molecule, localized within the nucleoid - the lightly staining region of the otherwise featureless prokaryotic cell. As well as this single ‘chromosome’, prokaryotes may also have additional genes on independent smaller, circular or linear DNA molecules called plasmids. However, this traditional view of the prokaryotic genome has been biased by the extensive research on E. coli, which has been accompanied by the mistaken assumption that E. coli is a typical prokaryote. In fact, prokaryotes display a considerable diversity in genome organization, some having a unipartite genome, like E. coli, but others being more complex. Borrelia burgdorferi B31, for example, has a linear chromosome of 911 kb, accompanied by 17 or 18 linear and circular molecules, which together contribute another 533 kb. Multipartite genomes are now known in many other bacteria and archaea.


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Eukaryotes

Animals, plants, fungi, and protists are eukaryotes, organisms whose cells are organized into complex structures enclosed within membranes. The defining membrane-bound structure which differentiates eukaryotic cells from prokaryotic cells is the nucleus.

Eukaryotic genomes

All of the eukaryotic nuclear genomes that have been studied are divided into two or more linear DNA molecules, each contained in a different chromosome. All eukaryotes also possess smaller, usually circular, mitochondrial genomes. Plants and other photosynthetic organisms possess a third genome, located in the chloroplasts or plastids.

There is a large amount of DNA which does not code for proteins and which varies widely in amount between species. In vertebrates only around 10% of the genome codes for proteins (exons) - much of the remaining 90% has no obvious function and may in fact be simply junk (introns). The differences in genome sizes within a class of organisms are almost entirely due to differences in the amount of this non protein coding DNA. Much of the non protein coding DNA is highly repetitive. The repetitiveness of DNA can be used as a way of classifying the different types of DNA which make up the eukaryotic genome.

Figure 4 Basic structures of eukaryotic and prokaryotic cells


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THE CENTRAL DOGMA OF MOLECULAR BIOLOGY

The central dogma of molecular biology states that the flow of genetic information is “DNA to RNA to PROTEIN”

There are four major stages represented by this dogma.

1. During replication, the DNA replicates its information with the use of several enzymes.

2. Transcription is the synthesis of a single-stranded RNA from a double-stranded DNA template. RNA synthesis occurs in a 5’→ 3’ direction and its sequence corresponds to that of the DNA strand, which is known as the sense strand.

3. In eukaryotic cells, the messenger RNA (mRNA) synthesised during transcription is processed by splicing and migrates to the cytoplasm.

4. During translation, the processed mRNA carries coded information to ribosomes in the cytoplasm. The ribosomes then read the information on the mRNA template and link amino acids in the prescribed order.

For the purposes of this course, the section on proteins will be excluded; however a short description on translation will be given before the conclusion of this chapter.

Figure 5 In vivo transcription of DNA to RNA and the translation of RNA to protein

(Adapted from http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html )

http://www.emc.maricopa.edu/faculty/farabee/BIOBK/BioBookPROTSYn.html


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DNA REPLICATION

DNA replication is the process of duplicating the DNA sequence in the parent strand to produce an exact replica (daughter strand). Replication is semi-conservative: each one of the two parental strands serves as a template for the new strand synthesis; therefore, duplicated double helices contain one parental strand and one daughter strand. DNA polymerases are the enzymes responsible for DNA synthesis. These enzymes use a single-stranded DNA template to catalyze the polymerization of a complementary DNA strand.

In a cell, DNA replication must happen before cell division can occur. DNA synthesis begins at specific locations in the genome, called "origins", where the two strands of DNA are separated. RNA primers attach to single stranded DNA and the enzyme DNA polymerase extends the primers to form new strands of DNA, adding nucleotides matched to the template strand. The unwinding of DNA and synthesis of new strands forms a replication fork. In addition to DNA polymerase, a number of other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis.

DNA replication can also be performed artificially, using the same enzymes used within the cell. DNA polymerases and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs artificial synthesis in a cyclic manner to rapidly and specifically amplify a target DNA fragment from a pool of DNA.

The replication process in bacteria

The chromosome of a prokaryote is a circular molecule of DNA. Replication begins at one origin of replication and proceeds in both directions around the chromosome. The basic steps of DNA replication are:

• Initiation – replication begins at an origin of replication

• Elongation – new strands of DNA are synthesized by DNA polymerase

• Termination – replication is terminated differently in prokaryotes and eukaryotes

Initiation

This step involves the assembly of a replication fork (bubble) at an origin of replication sequence of DNA found at a specific site of the circular chromosome of a bacterium. This origin of replication is unwound, and the partially unwound strands form a "replication bubble", with one replication fork on either end. Each group of enzymes at the replication fork moves away from the origin, unwinding and replicating the original DNA strands as they proceed. The factors involved are collectively called the pre-replication complex. It consists of the following:


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• A helicase, which unwinds and splits the DNA ahead of the fork. Thereafter, single-strand binding proteins (SSB) swiftly bind to the separated DNA, thus preventing the strands from reuniting. Because DNA is helical, this unwinding means that the DNA needs to rotate to avoid too much supercoiling. An enzyme, named DNA topoisomerase I, precedes the replication complex, and cleaves one strand of DNA ahead of the replication machinery, allowing free rotation of the DNA between the nick and the replication complex.

• A primase (an RNA polymerase), which generates an RNA primer that serves as a starting point or primer for synthesis of the new DNA chain.

• A DNA polymerase III holoenzyme, which in reality is a complex of enzymes that together perform the actual replication.

Elongation

After the helicase unwinds the DNA, single-strand binding protein is used to hold the DNA strands apart. RNA primase is then bound to the starting DNA site, and it synthesizes an RNA primer complementary to the DNA.

At the beginning of replication, an enzyme called DNA polymerase III holoenzyme binds to the RNA primer, which indicates the starting point for the replication.

• DNA polymerase can only synthesize new DNA from the 5’ to 3’ (of the new DNA - i.e. it moves on a template from 3’ to 5’). Because of this, the DNA polymerase can only travel on one of the original DNA strands without any interruption. This original strand, which goes from 3’ to 5’, is called the leading strand (Fig. 17). The complement of the leading strand, from 5’ to 3’, is the lagging strand.

• The holoenzyme catalyses the formation of a phosphodiester bond between the 3’-OH group of the last sugar on a chain of DNA (the primer), and the 5’ phosphate group on an incoming nucleotide triphosphate, chosen for its complementarity to the facing nucleotide present on the template strand (the one being copied).


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Figure 6 The replication fork

Replication of the lagging strand is more complicated than that of the leading strand. The orientation of the lagging strand is opposite to the working orientation of DNA polymerase III (which can only synthesize new DNA from the 5’ to 3’). As a result, the DNA of the lagging strand is replicated in a piecemeal fashion. The primase, which accompanies the holoenzyme, synthesizes RNA primers along the lagging strand every few hundreds of base pairs, which are then used as primers for DNA polymerase III action. Small stretches of DNA (so-called Okazaki fragments, ~2 kb), are synthesized until the next RNA primer which prevents DNA polymerase III activity. Then DNA polymerase I enters into action, digesting the RNA primer, and replacing ribonucleotides with deoxyribonucleotides. Finally DNA ligase generates the last phosphodiester bond between two newly synthesized Okazaki fragments.

Figure 7 DNA replication in bacteria (Taken from: http://commons.wikimedia.org/wiki/Image:DNA_replication_editable.svg )

http://commons.wikimedia.org/wiki/Image:DNA_replication_editable.svg


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DNA Helicase is an enzyme that unravels the DNA double helix and breaks the hydrogen

bonds.

DNA primase is an enzyme that generates an RNA sequence that serves as a starting point or

primer for synthesis of the new DNA chain.

DNA polymerases are enzymes that synthesize a complementary DNA strand using the original

strand as a template. In DNA synthesis, the new strand grows 5' to 3'.

An Okazaki fragment is a stretch of non-parental DNA produced along the lagging strand of

parental DNA by the DNA polymerase beginning at primer.

Three DNA polymerases are found in bacteria 1) DNA polymerase I (Pol I) functioning essentially in DNA repair, and a little in DNA replication.

DNA polymerase I possesses three enzymatic activities:

• 5' to 3' (forward) DNA polymerase activity, requiring a 3' primer site and a template

strand. DNA polymerase I catalyzes the addition of nucleotides to the 3’ hydroxyl of

primer DNA and requires dNTPs and Mg2+. Synthesis is always in the 5’ to3’ direction.

• A 5’ to 3’ (forward) exonuclease activity that mediates “nick translation” during DNA

repair.

• A 3’ to 5’ (reverse) exonuclease activity that allows “proofreading” and mediates

removal of RNA primers during replication.

2) DNA polymerase II (Pol II) is a DNA repair enzyme involved in replication of damaged DNA.

It has 3' to 5' exonuclease activity that mediates proofreading. DNA polymerase II differs from

DNA polymerase I in that it lacks 5' to 3' exonuclease activity and cannot use a nicked duplex

template.

3) DNA polymerase III (Pol III) is the main enzyme used in DNA replication; it is a complex of

several proteins (at least 20), forming the so-called holoenzyme. DNA polymerase III has 5' to 3'

(forward) DNA polymerization activity and catalyzes the addition of dNTPs to the end of a new

DNA strand with release of inorganic pyrophosphate (PPi). It also has 3' to 5' exonuclease

proofreading capability. The holoenzyme has high processivity, i.e. the number of nucleotides

added per binding event. The rate of DNA synthesis is extremely fast: 30-60,000 nucleotides per

minute.


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Termination

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. Termination of DNA replication in E. coli is regulated through the use of termination sequences (Ter sites) and the Tus protein (terminator utilization substance protein). The Tus protein binds to the termination sites, these Tus -Ter complexes allow a replication fork to pass through in one direction, but not the other. Multiple Ter sites are located in the termination region of the E. coli chromosome and slow down and stop the movement of the replication forks in this region. As a result, the replication forks are constrained to always meet and terminate within the termination region of the chromosome.

Fidelity of DNA replication

The error rate in DNA replication is very low and has been estimated at 1 error in 10 million bases. DNA polymerase III proofreads the newly made strand before continuing with replication. When an incorrect nucleotide is incorporated, the 3’ end will be frayed. The DNA polymerase III recognizes the problem, "backs up" and removes the incorrect nucleotide by means of its 3' to 5' exonuclease activity. The correct nucleotide is then added to the chain and elongation is resumed.

Note: DNA polymerase III present in the replication fork has three important properties:

(i) Chain elongation (ii) Processivity and (iii) Proofreading

Catalytic activities of DNA polymerases:

Important note: DNA polymerases I, II and III carry both a 5’ to 3’ polymerase activity, and a 3’ to 5’ exonuclease activity which eliminates from an elongating chain of DNA any misincorporated (non complementary) nucleotides. This is known as proofreading activity.

• 5' to 3' polymerase activity for DNA synthesis (DNA Pol I, II and III)

• 5' to 3' exonuclease activity for removal of the DNA/RNA primer

• (allows DNA Pol I to destroy a strand of DNA or RNA located ahead of it (3’ to it) on the DNA template).

• 3' to 5' exonuclease activity for proofreading (DNA Pol I, II and III)

The replication process in viruses

Viral populations do not grow through cell division, because they are acellular; instead, they use the machinery and metabolism of a host cell to produce multiple copies of themselves. In many ways the


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replication is very similar to that of bacteria but the expression of virus genetic information is dependent on the structure of the genome of the particular virus concerned. In every case, the genome must be recognized and expressed using the mechanisms of the host cell.

The replication process in Eukaryotes

The DNA replication process in eukaryotes is essentially the same as in prokaryotes, with some differences. While replication begins at ori C in prokaryotes, there are multiple origins of replication in eukaryotes due to the sheer size of the chromosomes. Replication begins at specific places on the chromosome - the origin or "ori" region and proceeds bidirectionally.

As in prokaryotes, the two parent strands are unwound with the help of DNA helicases. Stabilizing proteins attach to the unwound strands, preventing them from winding back together. DNA polymerases α and δ are responsible for DNA synthesis in eukaryotes. DNA polymerase α begins with an RNA primer and then adds a few DNA nucleotides. DNA polymerase δ takes over and synthesizes DNA nucleotides at approximately 100 times the rate of DNA polymerase α. RNA primers, needed repeatedly on the lagging strand to facilitate synthesis of Okazaki fragments, are synthesized by subunits of DNA polymerase α. DNA Polymerases β and ξ are presumed to be involved in DNA repair in eukaryotes which may indicate that they are involved in RNA primer removal. Finally, as in prokaryotes, each new Okazaki fragment is attached to the completed portion of the lagging strand in a reaction catalyzed by DNA ligase.

Eukaryotic nuclear chromosomes are packaged by histone proteins into a condensed structure called chromatin. The replication of eukaryotic chromosomes presents additional problems, including the need to remove, replicate and replace histones and other similar proteins associated with the DNA double helix. In addition, eukaryotes must be able to deal with the linear ends of each chromosome arm (the telomeres), i.e. once the RNA primer is removed at the end of each arm of a chromosome, there is no 3’-OH end for the DNA polymerase to recognize and use to replace missing nucleotides. This function is accomplished by telomerase, an enzyme that adds specific DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3’ end of DNA strands in the telomere regions. This enzyme is a reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres.


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Figure 8 The replication of DNA in Eukaryotes

TRANSCRIPTION

Transcription is the synthesis of RNA under the direction of DNA. Messenger RNA (mRNA) is synthesized by transcription or copying of DNA, a process similar to DNA replication. The DNA sequence is copied by RNA polymerase to produce a complementary RNA strand, called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell. Other types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA are not necessarily translated into an amino acid sequence.

Unlike DNA replication, transcription does not need a primer to start. RNA polymerase simply binds to the DNA and, along with other cofactors, unwinds the DNA to create an initiation bubble and the bases on the two strands are exposed. But how does the RNA polymerase know where to begin? The starting point of a gene is marked by a certain base sequence which is called a promoter site. In prokaryotes, transcription begins with the binding of RNA polymerase to the promoter on the DNA molecule. At the start of initiation, the RNA polymerase is associated with a sigma factor that aids in finding the appropriate additional “signaling” base pairs downstream of the promoter sequences. Transcription initiation is far more complex in eukaryotes, the main difference being that eukaryotic RNA polymerases do not directly recognize their promoter sequences. In eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription.

As in DNA replication, RNA is synthesized in the 5' to 3' direction (from the point of view of the growing RNA transcript). Only one of the two DNA strands is transcribed into mRNA (remember that RNA is a single-stranded molecule), unlike DNA replication, where both strands are copied. The DNA strand that is


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transcribed is called the template strand (also known as the antisense strand), while its complement is called the informational strand (also called the coding or sense strand).

Since the template strand and the informational strand are complementary, and since the template strand and the mRNA molecule are also complementary, it follows that the messenger RNA molecule produced during transcription is a copy of the DNA informational strand. Unlike DNA replication, mRNA transcription can involve multiple RNA polymerases on a single DNA template and multiple rounds of transcription resulting in amplification of a particular mRNA, i.e. many mRNA molecules can be produced from a single copy of a gene. This step also involves a proofreading mechanism that can replace incorrectly incorporated bases.

In bacteria, just as there is a sigma factor to help signal the beginning of a gene, another factor called "rho" aids in terminating the process of transcription. When the end of the gene is near, the rho factor binds to the mRNA, destabilizing the interaction between the template and the mRNA. This releases the newly synthesized mRNA from the elongation complex, thus stopping transcription. An alternative strategy for transcription termination in bacteria is known as rho-independent transcription termination. RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop, followed by a run of Us, which makes it detach from the DNA template. Transcription termination in eukaryotes is less well understood. It involves cleavage of the new transcript, followed by template-independent addition of As at its new 3' end, in a process called polyadenylation.

The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. A transcription unit that is translated into protein contains sequences that direct and regulate protein synthesis in addition to coding the sequence that is translated into protein. The regulatory sequence that is before, or 5', of the coding sequence is called the 5' untranslated region (5’UTR), and the sequence found following, or 3', of the coding sequence is called the 3' untranslated region (3’UTR). Transcription has a lower copying fidelity than DNA replication since, although there are some proofreading mechanisms, they are fewer and less effective than the controls for copying DNA.


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Figure 9 Transcription of RNA from DNA

(Adapted from: http://biologysemester58.wikispaces.com/Molecular+Genetics )

Prokaryotes

Prokaryotic transcription occurs in the cytoplasm alongside translation. Prokaryotes do not have exons and introns and an RNA molecule corresponding to the DNA molecule is produced by RNA transcription. In prokaryotes, mRNA is not modified.

Eukaryotes

Eukaryotic transcription occurs in the nucleus, where it is separated from the cytoplasm by the nuclear membrane. The mRNA transcript is then transported into the cytoplasm where translation occurs. Eukaryotic DNA is wound around histones to form nucleosomes and packaged as chromatin. Chromatin has a strong influence on the accessibility of the DNA to transcription factors and the transcriptional machinery including RNA polymerase.

In most mammalian cells, only 1% of the DNA sequence is copied into a functional RNA (mRNA). Eukaryotic mRNA is modified through RNA splicing, 5' end capping, and the addition of a polyA tail. One of the most important stages in RNA processing is RNA splicing. In many genes, the DNA sequence coding for proteins, or "exons", may be interrupted by stretches of non-coding DNA, called "introns". In

http://biologysemester58.wikispaces.com/Molecular+Genetics


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the cell nucleus, the DNA that includes all the exons and introns of the gene is first transcribed into a complementary RNA copy called "nuclear RNA," or nRNA. In a second step, introns are removed from nRNA by a process called RNA splicing. The edited sequence is called "messenger RNA," or mRNA. Only one part of the DNA is transcribed to produce nuclear RNA, and only a minor portion of the nuclear RNA survives the RNA processing steps.

The mRNA leaves the nucleus and travels to the cytoplasm, where it encounters cellular bodies called ribosomes. The mRNA, which carries the gene's instructions, dictates the production of proteins by the ribosomes in a process known as translation.

TRANSLATION

Translation is the process of converting the mRNA sequence into an amino acid sequence. It occurs in the cytoplasm where the ribosomes are located. Ribosomes are made of a small and a large subunit which surround the mRNA. In translation, an mRNA sequence is used by the ribosome as a template to guide the synthesis of a chain of amino acids.

DNA transfers information to mRNA in the form of a code defined by the sequence of nucleotide bases. Since DNA and RNA are constructed from four types of nucleotides, there are 64 possible triplet sequences or codons (4x4x4); many more than the 20 needed to specify the common amino acids present in nature. Three of the possible codons specify the termination of the polypeptide chain. They are called "stop codons". That leaves 61 codons to specify only 20 different amino acids. Therefore, most of the amino acids are represented by more than one codon. The genetic code is therefore said to be degenerate.

The vast majority of genes are encoded with exactly the same code, known as the canonical genetic code, or simply the genetic code. In fact there are many variant codes; so it should be noted that the canonical genetic code is not universal. For example, in humans, protein synthesis in mitochondria relies on a genetic code that varies from the canonical code.

During protein synthesis, a ribosome moves along an mRNA molecule from the 5' end to the 3' end and "reads" its sequence three nucleotides at a time (codon). Each amino acid is specified by the mRNA's codon, which pairs with a sequence of three complementary nucleotides (anticodon) carried by a particular transfer RNA (tRNA) molecule. The other end of the tRNA has the amino acid attached to the 3'-OH group via an ester linkage. A tRNA molecule with an attached amino acid is said to be "charged".


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Figure 10 Structure of the charged transfer RNA (tRNA) molecule

When a small subunit of a ribosome charged with a tRNA + methionine (initiator tRNA) encounters an mRNA, it attaches and starts to scan for a start signal or start codon (AUG). When it finds the start sequence AUG, the codon for methionine, the large subunit joins the small one to form a complete ribosome and protein synthesis is initiated. A new charged tRNA (tRNA + amino acid) enters the ribosome, at the next codon downstream of the AUG codon. If its anticodon matches the mRNA codon it binds and the ribosome can link the two amino acids together (Note: if a tRNA with the wrong anticodon (and therefore the wrong amino acid) enters the ribosome, it cannot bind with the mRNA and is rejected). The ribosome then moves one triplet forward and a new charged tRNA can enter the ribosome and the procedure is repeated. When the ribosome reaches one of three stop codons, UAG, UAA or UGA, there are no corresponding tRNAs to that sequence. Instead termination proteins bind to the ribosome and stimulate the release of the polypeptide chain (the protein), and the ribosome dissociates from the mRNA.

Figure 11 Process of DNA translation

(Adapted from http://www.duke.edu/web/MAT/jennifer_sohn/unit/translation_oh.htm)


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REFERENCES

The relevant websites as indicated in the course outline:

1. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes

2. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html

3. http://en.wikipedia.org/wiki/DNA_replication

4. http://en.wikipedia.org/wiki/DNA_replication

5. http://www.biochem.ucl.ac.uk/bsm/prot_dna/family_descriptions/1ecr_single/1ecr_single.html

6. http://virology-online.com/general/Replication.htm

A few general references:

1. PROMEGA: Protocols and applications guide. 1996

2. HYDE, J 1990. Molecular parasitology. Van Nostrand Reinhold. New York

3. BROWN, T. A 1986. Gene cloning. Van Nostrand Reinhold (UK) Co. Ltd.

4. BROWN, T. A 1995 3rd Edition. Gene cloning – An introduction. Chapman & Hall, London.

5. D’AQUILA, R.T., BECHTEL, L.J.,VITELER, J. A., ERON, J.J., GORCZYCZ, P. and KAPLIN, J.C.,

1991. Maximizing sensitivity and specificity of PCR by preamplification heating. Nucleic Acids Res.

19: 3749.

6. DIEFFENBACH, C. W. and DVEKSLER, G. S. 1995. PCR primer. A laboratory manual. CSHL

Press.

7. EHRLICH, H.A, GELFAND, D. and SNINSKY, J.J., 1991. Recent advances in the polymerase

chain reaction. Science 252: 1643 – 1651.

8. KWOK, S. and HIGUCHI, R 1989. Avoiding false positives with PCR. Nature 339: 237 – 238.

9. LONGO, M. C., BERNINGER, M.S. and HARTLEY J.L. 1990. Use of uracil DNA glycosylase to

control carryover contamination in polymerase chain reactions. Gene 93: 125 – 128.

10. MAXAM, A. M and GILBERT, W 1980. Sequencing end-labelled DNA with base-specific chemical

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