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Topic 7: HL IB Biology

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DNA structure suggested a mechanism for DNA replication
Franklin's x-ray diffraction experiments demonstrated that the DNA helix is both tightly packed and regular in structure. Phosphates (and sugars) form an outer backbone and nitrogenous bases are packaged within the interior

Chargaff had also demonstrated that DNA is composed of an equal number of purines (A + G) and pyrimidines (C + T). This indicates that these nitrogenous bases are paired (purine + pyrimidine) within the double helix. In order for this pairing between purines and pyrimidines to occur, the two strands must run in antiparallel directions

When Watson & Crick were developing their DNA model, they discovered that an A-T bond was the same length as a G-C bond. Adenine and thymine paired via two hydrogen bonds, whereas guanine and cytosine paired via three hydrogen bonds. If the bases were always paired this way, then this would describe the regular structure of the DNA helix (shown by Franklin)

Consequently, DNA structure suggests two mechanisms for DNA replication:
- Replication occurs via complementary base pairing (adenine pairs with thymine, guanine pairs with cytosine).
- Replication is bi-directional (proceeds in opposite directions on the two strands) due to the antiparallel nature of the strands
Nucleosomes help to supercoil the DNA
One difference between eukaryotic DNA and bacterial DNA is that eukaryotic DNA is associated with histone proteins. Prokaryotes have naked DNA. Histones are used by the cell to package the DNA into structures called nucleosomes.

Nucleosomes consist of a central core of 8 histone proteins with DNA coiled around he proteins. The 8 proteins (octamer) consist of two copes of 4 different types of histones. A short section of "linker" DNA connects one nucleosome to the next. An additional histone protein molecule, called H1, serves to bind the DNA to the core particle. Supercoiling allows a great length of DNA to be packed into a much smaller space within the nucleus. The nucleosome is an adaption that facilitates the packing of the large genomes that eukaryotes possess. H1 histone binds in such a way to form a structure called the 30nm fibre that facilitates further packing
DNA replication is continuous on the leading strand and discontinuous on the lagging strand
Because the two strands of the DNA double helix are arranged in an anti-parallel fashion, synthesis on the two strands occurs in very different ways. Once strand, the leading strand, is made continuously following the fork as it opensThe lagging strand is made in fragments, moving away from the replication fork. New fragments, Okazaki fragments, are created on the lagging strand as the replication fork exposes more of the template strand
DNA replication is carried out by a complex system if enzymes
Replication involves the formation and movement of the replication form and synthesis of the leading and lagging strands.
Proteins are involved as enzymes at each stage but also have other functions

DNA gyrase = untwists
DNA helicase = separates
SSMP = attach after helicase has split DNA. Stops the DNA from immediately joining again after helicase has moved.
DNA polymerase 3 adds complementary base pairs
DNA Ligase adds the okazaki fragments

One side called leading strand has the CBP built no drama
Other side, lagging, RNA primase
DNA gyrase
Relaxes positive supercoils to prepare for uncoiling.
DNA helicase
Unwinds the DNA at the replication fork by breaking the hydrogen bonds between the bases on each strand



Before DNA replication can occur, the strand of the molecule must separate so they can act as a template for the formation of a new strand.
• This separation is carried out by helicases, a group of enzymes that use energy from ATP to break hydrogen bonds between complementary bases.
• One well-studied helicase consists of six globular polypeptides arranged in a donut shape. The polypeptides assemble with one strand of the DNA molecule passing through the centre of the donut and the other outside it.
• Energy from ATP is used to move the helicase along the DNA molecule, breaking Hbonds between bases and parting the two strands.
SSbP
Single stranded binding proteins keep the strands apart long enough to allow the template strand to be copied.
DNA primase
The RNA primer is necessary to initiate the activity of DNA polymerase. On the lagging strand there are a number of primers but on the leading strand there is just one. The enzyme DNA primase creates these RNA primers.
DNA polymerase
DNA polymerase is responsible for covalently linking the deoxyribonucleotide monophosphate to the 3' end of the growing strand. Different organisms have different kinds of DNA polymerases, each with different functions such as proof-reading, polymerization and removal of RNA primers once they're no longer needed.



Once helicase has unwound the double helix and split DNA, replication can begin.
Each of the two strands acts as a template for the formation of a new strand. Assembly of these new strands is carried out by the enzyme DNA polymerase.
DNA polymerase always moves along the template strand in the same direction, adding one nucleotide at a time.
Free nucleotide with each of the four possible bases are available in the area where DNA is being replicated. . DNA polymerase brings nucleotides into the position where hydrogen bond could form, but unless this happens and a complementary base pair is formed the nucleotide breaks away.

Once a nucleotide with the correct base has been brought into position and hydrogen bonds have been formed between the two bases, DNA polymerase links it to the end of the new strand by making a covalent bond between the phosphate group of the free nucleotide and the sugar of the nucleotide at the existing end of the other strand. The pentose sugar is 3' terminal and phosphate 5' terminal, so DNA polymerase adds on the 5' terminal of he free nucleotide to the 3' terminal of the existing strand.

DNA polymerase gradually moves along the template strand, assembling the new strand with a base sequence complementary to the template strand. It does this with a high degree of fidelity (low mistakes).
DNA ligase
DNA ligase connects gaps between fragments (links Okazaki fragments)
DNA Polymerases can only add nucleotides to the 3' end of a primer
DNA replication begins at sites called origins of replication.
In prokaryotes there is one site and in in eukaryotes there are many.
Replication occurs in both directions away from the origin. The result appears as a replication bubble in electron micrographs.

The phosphate group of new DNA nucleotides is added to the 3' carbon of the deoxyribose of the nucleotide at the end of the chain. Replication therefore occurs in the 5' to 3' direction
Some regions of DNA do not code for proteins but have other important functions.
Forming between 50 and 60% of the genome, repetitive sequences, which are especially common in eukaryotes, occur in two types: moderately repetitive sequences and highly repetitive sequences (satellite DNA).
Telomeres
Telomeres, an area of repetitive sequences occurring at the end of eukaryotic chromosomes, serve a protective function. During interphase, the enzymes that replicate DNA cannot continue replication all the way to the end of the chromosome, thus if cells went through the cell cycle without telomeres they would lose the genes at the end of the chromosomes.
Rosalind Franklin
When a beam of X-rays is directed at a material most of it passes through, however some is scattered by the particles in the materials, which is known as diffraction. The wavelength of X-rays makes them particularly sensitive to diffraction by the particles in biological molecules including DNA. The molecules of DNA in Franklin's samples were arranged in an orderly enough array for a diffraction pattern to be obtained, rather than random scattering. The sample can be rotated in three different dimensions to investigate the pattern of scattering, which can be recorded using X-ray film. Franklin developed a high-resolution camera to obtain very clear images of diffraction patterns from DNA.
Results of RF
Franklin deduced that the molecule was helical in shape due to the cross in the centre, the angle of the cross shape showed the pitch (steepness of angle) of the helix, the distance between horizontal bars showed turns of the helix to be 3.4nm apart, the distance between the middle of the diffraction pattern and the top showed there was a repeating structure within the molecule (0.34nm between the repeats), which was later found to be the vertical distance between adjacent base pairs in the helix.
The Sanger Method. Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing.
In the Sanger method, many copies of the unknown DNA that is to be sequenced are placed into test tubes with all the raw materials, including deoxyribonucleotides and the enzymes necessary to carry out replication. Very small quantities of dideoxyribonucleotides that have been labelled with different fluorescent markers, which will stop the replication at precisely the point they were incorporated into the new DNA. The fragments are separated by length using electrophoresis. The sequence of bases can be automatically analysed by comparing the colour of the fluorescence with the length of the fragment.
DNA profiling
A variable number tandem repeat (VNTR) is a short nucleotide sequence that can be inherited as an allele which shows variations between individuals in terms of the number of times the sequence is repeated. Analysis of VNTR allele combinations in individuals is the basis of DNA profiling, which has applications in genealogical investigations. Genealogists deduce paternal lineage by analysing short tandem repeats from the Y chromosome and deduce maternal lineage by analysing mitochondrial DNA variation in single nucleotides at specific locations called hypervariable regions.
Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing.
• The determination of the sequence of bases in a genome is carried out by most commonly using a method that employs fluorescence.
• Many copies of the unknown DNA that is to be sequences are placed into test tubes with all the raw materials including deoxyribonucleotides and the enzymes necessary to carry out replication.
• In addition very small quantities of dideoxyribonucleotides that have been labelled with different fluorescent markers are added.
• The dideoxyribonucleotides will be incorportated into some of the new DNA, but when they are incorporated they will stop the replication at precisely the point where they were added.
• The fragments are separated by length using electrophoresis.
• The sequence of bases can be automatically analysed by comparing the colour of the fluorescence with the length of the fragment.
Hershey and Chase
From the late 1800s, scientists were convinced chromosomes, consisting of both protein and nucleic acid, were involved in inheritance. The view that protein was the hereditary material was favoured as it was a macromolecule that had great variety (20 naturally occurring sub-units) and had been identified to have many specific functions. Alfred Hershey and Martha Chase were able to ascertain whether protein or DNA was the genetic material of viruses. By the 1950s, viruses were known to be infectious particles that bind to host cells and inject their genetic material whilst the non-genetic portion remains outside causing the infected cell to manufacture large numbers of new viruses and then burst to release the viruses. They used T2 bacteriophage because of its simple structure consisting of a coat composed of protein while DNA is found inside the coat.• They cultured the viruses to contain proteins with radioactive sulphur and they separately cultured viruses that contained DNA with radioactive phosphorus.
• They infected bacteria separately with the two types of viruses
• They used a blender to separate the non-genetic component of the virus from the cell and then centrifuged the culture solution o concentrate the cells in a pellet.
• The cells were expected to have the radioactive genetic component of the virus in them.
• They measured the radioactivity in the pellet and the supernatant.
Utilization of molecular visualization software to analyse the association between protein and DNA within a nucleosome
1. The molecule has two copies of each histone protein, identified by the tails that extend from the core.
2. Approximately 150bp of DNA wrapped around nearly twice around the octamer core
3. N-terminal tail that projects from the histone core for each protein. Chemical modification of this tail is involved in regulating gene expression
4. Visualize the positively charged amino acids on the nucleosome core.
Purines
2 rings (adenine and guanine)
Pyrimidine
1 ring (Thymine and cytosine)
...
The synthesis of mRNA occurs in three stages: initiation, elongation and termination. Transcription begins near a site in the DNA called the promoter. Once binding of the RNA polymerase occurs, the DNA is unwound by the RNA polymerase forming an open complex. The RNA polymerase slides along the DNA, synthesising a single strand of RNA.
Chemical modification of histones
Chemical modification of the tails of histones, which are proteins associated with eukaryotic DNA, is an important factor in determining gene expression. Modification can involve the addition of an acetyl group, methyl group or phosphate group. For example, normally the residues of the amino acid lysine on histone tails, which can have either acetyl groups removed or added, bear a positive charge that can bind to the negatively charged DNA to form a condensed structure that inhibits transcription. Histone acetylation neutralises these positive charges allowing a less condense structure with higher levels of transcription. Chemical modification of histone tail can either activate or deactivate genes by decreasing or increasing the accessibility of the gene to transcription factors.
Eukaryotic cells modify mRNA after transcription:
Transciption, translation and post-translational regulation occur in both eukaryotes and prokaryotes. However most regulation of prokaryotic gene expression occurs at transcription and the method of gene expression of post-transcriptional modification of RNA does not occur in prokaryotes. The absence of a nuclear membrane surrounding the genetic material in prokaryotes means that transcription and translation can be coupled. The separation of the location of transcription and translation in eukaryotes allows significant post-transcriptional modification to occur before the mature transcript exists the nucleus. For example, the removal of intervening sequences (introns) from the RNA transcript. Prokaryotic DNA does not contain introns. The immediate product of mRNA transcription, prior to post-transcriptional modification, is known as pre-mRNA.Interspersed throughout the mRNA are sequences that do not contribute to the formation of the polypeptide, intervening sequences or introns, which must be removed and the remaining coding portion of the mRNA, exons, must be spliced together in order to form mature mRNA. Post-transcriptional modification also includes the addition of a 5' cap that usually occurs before transcription has been completed. A poly-A tail is added after the transcript has been made.
Splicing of mRNA increases the number of different proteins an organism can produce:
Alternative splicing, occurring in genes with a multiple exons, is a process during expression whereby a single gene codes for multiple proteins. A particular exon may or may not be included in the final mRNA, thus the proteins translated from alternatively spliced mRNAs will differ in their amino acid sequence and possibly in biological functions. For example, in mammals the protein tropomyosin is encoded by a gene that had eleven exons and as result tropomyosin pre-mRNA is spliced differently in different tissues resulting in five different forms of the proteins (e.g. in skeletal muscle, exon '2' is missing from the mRNA and in smooth muscle, exons '3' and '10' are not present).
Regulation of gene expression in prokaryotes
Gene expression in prokaryotes results from variations in environmental factors. The genes responsible for the metabolism of lactose by E.coli are only expressed when lactose is present as it causes the deactivation of a repressor protein. The breakdown of lactose results in regulation by negative feedback. Once the lactose is broken down, the repressor protein is no longer deactivated and proceeds to block the expression of lactose metabolism genes.
When is gene expression regulated?
Whilst proteins that are always necessary for the survival of the organism are expressed in an unregulated fashion, however those that are produced at certain times and in certain amounts must be regulated.
Regulation of gene expression in eukaryotes
Eukaryotic genes are regulated in response to variation in environmental factors as each cell of a multicellular eukaryotic organisms expresses only a fraction of its genes. The regulation of eukaryotic gene expression is also part of cellular differentiation and the process of development. Enhancers, silencers and promotor-proximal elements are proteins which bind to DNA and regulates transcription.
Proteins that regulate eukaryotic gene expression
Unlike the promotor sequence, the sequences linked to regulatory transcription factors are unique to the gene. Enhancers are regulatory sequences on the DNA which increase the rate of transcription when proteins bind to them. Silencers are those sequences on the DNA which decrease the rate of transcription when proteins bind to them. While enhancers and silencers can be distant from the promoter, promoter-proximal elements are nearer to the promoter and binding of proteins to them is necessary to initiate transcription.
Effect of environmental factors on gene expression
The effect of environmental on gene expression is unequivocal for some traits, such as the production of skin pigmentation during exposure to sunlight in humans. In embryonic development, the uneven distribution of chemicals called morphogens in the embryo contributes to different patterns of gene expression and thus the different fates of the embryonic cells depending on their position in the embryo.
The 'C' gene in cats codes for the production of enzyme tyrosinase, the first step in the production of pigment. However a mutant allele, 'cs', allows normal pigment production only at temperatures below body temperature as the protein product is less active resulting in less pigment at higher temperatures. This allele has been selected for in the selective breeding of Siamese cats.
The promoter as an example of non-coding DNA with a function.
There are a number of non-coding sequences in the genome which do not code for the production of polypeptides, rather some of them have functions such as the production of tRNA and rRNA. Some non-coding regions play a role in the regulation of gene expression such as enhancers and silencers. The promoter is a sequence that is located near a gene and is the binding site of RNA polymerase, the enzyme that catalyses the formation of the covalent bond between nucleotides during the synthesis of DNA. The promoter is not transcribed, however is still involved in transcription.
Analysis of changes in the DNA methylation patterns.
Whereas methylation of histones can promote or inhibit transcription, direct methylation of DNA tends to increase gene expression. The amount of DNA methylation varies during a lifetime and is affected by environmental factors.
Initiation of Translation
To begin the translation process, an mRNA molecule binds to the small ribosomal subunit at an mRNA binding site. An initiator tRNA molecule carrying methionine then binds at the start codon 'AUG'. The large ribosomal subunit then binds to the small one. The initiator tRNA is in the P site. The next codon signals another tRNA to bind, occupying the A site. A peptide bond is formed between the amino acids in the P and A site.
Elongation of Translation
Following initiation, elongation occurs through a series of repeated steps. The ribosome translocates three bases along the mRNA, moving the tRNA in the P site to the E site, freeing it and allowing a tRNA with the appropriate anticodon to bind to the next codon and occupy the vacant A site. The process continues until a stop codon is reached when the free polypeptide is released. The direction of movement along the mRNA is from the 5' end to the 3' end.
Use of Free Ribosomes
Proteins that are destined for use in the endoplasmic reticulum, the Golgi apparatus, lysosomes, the plasma membrane or outside the cell are synthesised by ribosomes bound to the endoplasmic reticulum. The presence of a signal sequence, which is the first part of the polypeptide translated, determines whether the ribosome is free in the cytosol or bound to the endoplasmic reticulum. As the signal sequence is created it becomes bound to a signal recognition protein that stops the translation until it can bind to a receptor on the surface of the endoplasmic reticulum. Once this occurs, translations begins again with the polypeptide moving into the lumen of the endoplasmic reticulum as it is created
Use of Bound Ribosomes
Proteins that are destined for use in the endoplasmic reticulum, the Golgi apparatus, lysosomes, the plasma membrane or outside the cell are synthesised by ribosomes bound to the endoplasmic reticulum. The presence of a signal sequence, which is the first part of the polypeptide translated, determines whether the ribosome is free in the cytosol or bound to the endoplasmic reticulum. As the signal sequence is created it becomes bound to a signal recognition protein that stops the translation until it can bind to a receptor on the surface of the endoplasmic reticulum. Once this occurs, translations begins again with the polypeptide moving into the lumen of the endoplasmic reticulum as it is created
Translation in eukaryotes vs. prokaryotes
Eukaryotes have a compartmentalised cell structure, whereas prokaryotes do not. Once transcription is complete in eukaryotes, the transcript is modified in several ways before existing the nucleus. Thus, there is a delay between transcription and translation due to compartmentalisation. In prokaryotes, as soon as the mRNA is transcribed, translation begins.
Primary structure of a protein
A chain of amino acids is called a polypeptide. There are 20 commonly occurring amino acids that can be combined in any sequences, thus there is a huge diversity of proteins. The sequence of amino acids in a polypeptide is termed its primary structure
Secondary structure of a protein
The chain of amino acids in a polypeptide has polar covalent bonds within its backbone, and thus tends to fold in such a way that hydrogen bonds form between the carboxyl (C=O) group of one residue and the amino group (N-H) group of an amino acid in another part of the chain. This results in the formation of patterns within the polypeptide called secondary structure, for example α helices and β pleated sheets.
Tertiary structure of a protein
Tertiary structure refers to the overall three-dimensional shape of the protein, which results from the interaction of R-groups with one another and with the surrounding water medium. There are several types of interaction:
- Positively charged R groups with negatively charged R groups
- Hydrophobic amino acids will orientate themselves toward the centre of the polypeptide to avoid contact with water whilst hydrophilic will orientate outwards
- Polar R groups form hydrogen bonds with other polar R groups
- The R group of cysteine can form a covalent bond with the R group of another cysteine forming a disulphide bridge.
Quaternary structure of a protein
Proteins can be formed from a single polypeptide chain (e.g. lysozyme) or from more than one polypeptide chain (e.g. insulin is made of two chains and haemoglobin is made of four chains). Quaternary structure refers to the way polypeptides fit together when there is more than one chain and the addition of non-polypeptide components. The biological activity of a protein is related to its primary, secondary, tertiary and quaternary structure. Factors such as high temperatures and changes in pH can cause alterations in the structure of a protein and therefore disrupting its biological activity, denaturing it.
tRNA-activating enzymes
Each tRNA molecule is recognised by a tRNA-activating enzyme that attaches a specific amino acid to the tRNA, using ATP for energy. The base sequence of tRNA molecules varies and this causes some variability in structure. Activation of a tRNA molecule involves the attachment of an amino acid to the 3' terminal of the tRNA by a tRNA-activating enzymes, of which there are twenty different types that are specific to one of the 20 amino acids and the correct tRNA molecule. The active site of the activating enzyme is specific to both the amino acid and tRNA. Once ATP and an amino acid are attached to the active site of the enzyme, the amino acid is activated by the formation of a bond between the enzyme and adenosine monophosphate (AMP). Then the activated amino acid is covalently attached to the tRNA. Energy from this bond is later used to link the amino acid to the growing polypeptide chain during translation.
Ribosome structure
Ribosome structure includes:
• Proteins and ribosomal RNA molecules (rRNA)
• Two sub-units, one large and one small.
• Three binding sites for tRNA on the surface of the ribosome ('E' - exit site, 'P' -peptidyl site and 'A' - aminoacyl site). Two tRNA molecules can bind at the same time to the ribosome.
• There is a binding site for mRNA on the surface of the ribosome.
Polysomes
Polysomes are structures that represent multiple ribosomes attached to a single mRNA molecule. Due to the fact that translation and transcription occur in the same compartment in prokaryotes and translation directly follows translation, multiple polysomes are visible associated with one gene. In eukaryotes, polysomes occur in both the cytoplasm and next to endoplasmic reticulum.
a cluster of ribosomes held together by a strand of messenger RNA which each is translating.