UNIT 7 – NUCLEIC ACIDS

Outline the features of DNA structure that suggested a mechanism for DNA replication.

1\. Complementary base pairing: A-T and C-G ensures that the sequence of the genetic code is conserved when each strand replicates. \n \n 2. Covalently bonded sugar-phosphate backbone: relatively strong bonds along the DNA backbone ensure the nucleotides of the parent strands remain in the same sequence during replication. \n \n 3. Hydrogen bonds between complementary strands: allow the parent DNA strands to easily separate to serve as templates for new strands during DNA replication.

Outline the levels of DNA packaging.

1. DNA double helix.
2. DNA wraps around histone proteins, forming nucleosomes and the "beads on a string" structure.
3. Multiple nucleosomes wrap into a fibre (chromatin).
4. Supercoiling of the chromatin produces the chromosome (during mitosis and meiosis).

Outline the process of X-ray diffraction.

An X-ray is shown on a solid crystal. The structure of that crystal causes the X-ray beam to diffract into many directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the atomic structure of the crystal.

Outline the role of dideoxynucleotides in the process of DNA sequencing.

The "chain-termination sequencing" method requires a single-stranded DNA template to be sequenced, a DNA primer, a DNA polymerase III, normal DNA nucleotides, and modified nucleotide called a di-deoxynucleotide triphosphates (ddNTPs). DNA replication proceeds until by chance the DNA polymerase III adds a ddNTP to an elongating strand. ddNTPs lack the 3'-OH group required for the formation of a bond between two nucleotides, causing DNA polymerase III to cease extension of DNA when a modified ddNTP is incorporated.

Outline the mechanism of histone-DNA association in a nucleosome.

Nucleosomes facilitate DNA packaging in a eukaryotic cell. A nucleosome is composed of **positively-charged histone proteins** that strongly adhere to the **negatively-charged DNA.** 

Outline the deductions about DNA structure made from the X-ray diffraction pattern.

Taken in 1952, this image is the first X-ray picture of DNA, which led to the discovery of its molecular structure. Created by Rosalind Franklin using a technique called X-ray diffraction, it revealed the **double helix shape** of the DNA molecule. In addition, the **radius**, **pitch**, **pitch angle**, and the **number of phosphate molecules per pitch** of the DNA helix could be determined. The pitch of a helix is the height of one complete helix turn

Outline the role of enhancers in the regulation of gene expression.

Many eukaryotic genes possess ***enhancer sequences***, which can be found at considerable distances from the genes they affect. Enhancer sequences are DNA sequences that, when bound by proteins called transcription factors, "enhance" (meaning activate or "turn on") the transcription of an associated gene. 

Explain the control of gene expression in eukaryotes.

Gene expression can be controlled by turning on/off genes at different times so only specific mRNA is produced through transcription. In eukaryotic cells, most genes are turned off (not being transcribed) at any one time. Gene expression is primary controlled by proteins called **transcription factors**, which bind to DNA to either increase or decrease transcription by enhancing or inhibiting the ability of RNA polymerase to bind to the promoter. 

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When DNA is coiled around histone proteins in a methylated **nucleosome**, the transcription factors can not bind to DNA. This will "silence" a gene so it will not be transcribed. 

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When acetylated, nucleosome will uncoil. This will expose the DNA, allowing transcription factors to bind. As a result, a genes will be accessible for transcription. 

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**Hormones** and **environmental chemicals** can interact with transcription factors to indirectly affect gene expression. For example, the plant hormone auxin interacts with transcription factors to regulate gene expression in plant meristems.

Outline two examples of environmental influence on gene expression.

The protein responsible for brown/black coloration in the skin and hair/fur of many animals, including humans, is called melanin, which is produced in a biological pathway regulated by the enzyme tyrosinase. Himalayan rabbits (and Siamese cats) carry "**temperature sensitive tyrosinase genes**" which controls fur pigmentation. The genes are expressed differently depending on temperature - the cooler the body, the darker the fur!

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The **development of a flower** by an angiospermatophyta is due to differentiation by cells in the shoot apex. The change in gene expression is in response to the change of light/dark cycle in the environment. Different genes are expressed in different regions of the tip of the shoot before, during and after formation of a flower.

Describe the initiation of transcription.

Initiation is the beginning of transcription. It occurs when the enzyme **RNA polymerase** binds to a region of the DNA upstream of the gene at a specialized sequence called the **promoter**. This signals the DNA to unwind so the enzyme can ''read'' the bases in one of the DNA strands. The enzyme is now ready to make a strand of mRNA with a complementary sequence of bases.

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**Transcription factors** are proteins that bind to enhancer or promoter regions of the DNA and interact to activate the transcription of a particular gene.

Compare gene expression between prokaryotic cells and eukaryotic cells.

Prokaryotic cells lack a cell nucleus, and their DNA therefore floats freely in the cell cytoplasm. To synthesize a protein, the processes of **transcription and translation occur almost simultaneously** within the **cytoplasm**.

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In eukaryotic cells, the DNA is contained inside the cell’s nucleus and there it is transcribed into RNA. The newly synthesized RNA is then transported out of the nucleus into the cytoplasm, where ribosomes translate the RNA into protein. The processes of **transcription and translation are physically separated by the nuclear membrane**; transcription occurs only within the nucleus, and translation occurs only outside the nucleus in the cytoplasm. Because they occur in different cellular locations, transcription and translation of a gene **do not occur simultaneously.** 

Describe the process of alternative RNA splicing.

Alternative splicing is a process in which exons from the same gene are joined in different combinations, leading to different mRNA transcripts. These mRNAs will be translated to produce different proteins with distinct structures and functions — all from a single gene. Alternative splicing increases the diversity of the proteins that can be coded from the genome.

Compare methylation patterns in twins using superimposed images of dyed chromosomes.

Because they have identical genomes, identical twins are ideal subjects for studying the effects of epigenetic modifications - changes to the DNA which control the expression of genes but not the DNA sequence itself. **Epigenetic modifications, such as DNA methylation, control which genes are switched ‘on’ and ‘off’ in a particular cell or tissue.** Epigenetic marks in the DNA can be altered by many different things, including environmental factors such as diet, exercise and smoking. 

Define “epigenome”

An **epigenome** consists of the chemical changes to the DNA (i.e. methylation) and histone proteins (i.e. acetylation) of an organism. These changes can be passed down to an organism's offspring, which goes against the idea that inheritance happens only through the DNA code that passes from parent to offspring. It means that **a parent's experiences, in the form of epigenetic tags, can be passed down to future generations**.

Discuss the role of "reprogramming" of DNA.

***Reprogramming*** is the removal of epigenic marks, such as DNA methylation, early in embryonic development (zygote to blastocyst). Essentially, the methyl groups on the maternal DNA (from egg) and paternal DNA (from sperm) are removed. This converts the DNA to a less-differentiated state. Egg and sperm are highly differentiated cell types, and with reprogramming the zygote and embryo become undifferentiated stem cells. 

Discuss the role of "imprinting" of DNA.

Humans inherit two alleles from mother and father. Both are functional for the majority of the genes, but sometimes one is turned off and isn't expressed in offspring.

Imprinting is the process by which only one copy of a gene in an individual (either from their mother or their father) is expressed, while the other copy is suppressed. Genomic imprinting does not affect the DNA sequence itself. Instead, gene expression is silenced by the epigenetic addition of chemical tags (usually methyl) to the DNA during egg or sperm formation. The imprinted **gene is silenced in the offspring because the DNA methylation was inherited from the parent egg or sperm.** DNA methylation keeps DNA tightly bound to the nucleosome, keeping transcription from occurring. Epigenetic tags on imprinted genes usually stay in place for the life of the individual.

Outline the process of translation termination, including the role of the stop codon.

(I) The ribosome moves down the mRNA until there is a stop codon in the A site. (II) No tRNA molecules can bind to stop codons. (III) Proteins called **release factors** bind to the A site. (IV) This causes: the polypeptide chain to be released from the tRNA in the P site. The ribosome seperates from the mRNA and splits into large and small subunits.

List destinations of proteins synthesized on free ribosomes.

The cytoplasm, mitochondria and chloroplasts.

List destinations of proteins synthesized on bound ribosomes.

The ER, the Golgi apparatus, lysosomes, the plasma membrane or outside the cell.

Outline how a ribosome becomes bound to the endoplasmic reticulum

(Whether the ribosome is free in the cytosol or bound to the ER depends on the presence of a signal sequence on the polypeptide being translated. It is the first part of the polypeptide translated). 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 ER. Once this happens, translation begins again with the polypeptide moving into the lumen of the ER as it is created.

State the three fates of proteins synthesised by ribosomes associated with the RER

1. To be secreted by exocytosis (e.g. antibodies.)
2. To be plasma membrane proteins (e.g. Voltage-gated potassium ion channels)
3. To function in membrane-bound organelles (e.g. digestive enzymes with lysosomes)

Explain how the chemical characteristics of R groups in the polypeptide chain affect protein folding.

If surrounded by water, amino acids with hydrophobic (non-polar) R groups tend to be located in the center of the protein and those with hydrophilic (polar or charged) R groups tend to be on the outside

Outline the process of attaching an amino acid to tRNA by the tRNA activating enzyme.

ATP is required to supply energy to link the tRNA with its specific amino acid via a covalent bond. The formation of this covalent bonds conserves energy (originally from ATP), which is then used to join amino acids via peptide bonds (at the ribosome). The reaction occurs in two steps.


1. the activation of the amino acid. ATP and the specific amino acid bind to the tRNA activating enzyme. ATP is hydrolysed and the amino acid is covalently linked to AMP.
2. the attachment of amino acid to tRNA. The specific tRNA molecule then binds to the active site. The amino acid is covalently linked to the tRNA and AMP is released. The charged tRNA (tRNA with amino acid) is released from the active site.

Outline the structure of tRNA molecules.

tRNA molecules have a stem-loop structure. The stem regions are double stranded due to hydrogen bonding between complementary base pairs. These are three loops, where there is no base pairing. One loop is called the anticodon loop and contains the three base anticodon sequence. There is an amino acid attachment site at the 3' end of the tRNA molecule.

Identify the beginning of an mRNA strand in a micrograph of polysomes

A **polysome** (or a polyribosome) is a group of two or more ribosomes translating an mRNA sequence simultaneously

* The polysomes will appear as beads on a string (each 'bead' represents a ribosome ; the ‘string’ is the mRNA strand)
* In prokaryotes, the polysomes may form while the mRNA is still being transcribed from the DNA template
* Ribosomes located at the 3’-end of the polysome cluster will have longer polypeptide chains that those at the 5’-end

Define bioinformatics.

The use of computers to store and analyse the huge amounts of data being generated by the sequencing of genomes and the identification of gene and protein sequences.

List seven species for which the entire genome has been sequenced.

humans, E.coli, yeast (S. cerevisiae), fruit flies (D. melangoaster), a soil roundworm (C. elegans), thale cress (A. thalania) and mice (M. musculus)