DNA, Genes and Genomics

Prokaryotic DNA The prokaryotes usually have only one chromosome, and it bears little morphological resemblance to eukaryotic chromosomes. Consists of single, circular DNA molecule located in the nucleoid region of cell. Referred to as being “naked” Bacterial cells may also contain multiple plasmids - small circular fragment of DNA separate from the main chromosome.

Eukaryotic DNA Structure DNA consists of two molecules that are arranged into a ladder-like structure called a Double Helix. A molecule of DNA is made up of millions of tiny subunits called Nucleotides. Each nucleotide consists of: Phosphate group Ribose sugar Nitrogenous base

Nucleotides Phosphate Nitrogenous Base Ribose Sugar

Nucleotides The phosphate and sugar form the backbone of the DNA molecule, whereas the bases form the “rungs”. There are four types of nitrogenous bases.

Nucleotides A Adenine T Thymine C Cytosine G Guanine

Nucleotides Each base will only bond with one other specific base. Adenine (A) Thymine (T) Cytosine (C) Guanine (G) Form a base pair. Form a base pair.

DNA Structure A C T G G A T C Because of this complementary base pairing, the order of the bases in one strand determines the order of the bases in the other strand.

DNA Replication unfolding and unwinding of the DNA double helix at hundreds of points, known as replication origins, along the chromosome. The enzyme helicase separates the two DNA strands, separating them like opening a zipper, with the point of opening being termed the replication fork. Where the DNA strands are separated, a short length of RNA binds to each DNA strand under the control of the enzyme, DNA primase. This RNA acts as a primer (see figure 11.26a page 405).

DNA Replication A DNA polymerase enzyme can then proceed to build new DNA strands using each of the old strands as a template (see figure 11.26b). Replication of DNA can occur only in the 5´ to 3´ direction. This is no problem with the so-called leading strand because its new complementary strand can be built continuously in the 5´ to 3´ direction. The other strand, known as the lagging strand, can be built only backwards and in short discontinuous pieces (Okasaki fragments - see figure 11.26b). When finished, the RNA primers are removed, the gaps are filled by another DNA polymerase and the pieces are joined by the enzyme, DNA ligase.

DNA Replication Watch DNAi clip

Mitochondrial DNA Mitochondria contain mtDNA, a double stranded circular molecule comprising: 16 568 base pairs and code for 37 genes: 13 genes code for proteins that are involved in cellular respiration 2 genes code for ribosomal RNA (rRNA) 22 genes code for transfer RNAs (tRNAs).

PROTEIN SYNTHESIS An individuals characteristics are determined by their DNA. The DNA determines which proteins are made. The most important proteins are enzymes. The sequence of bases in the DNA determines the sequence of amino acids in the protein. This is known as the GENETIC CODE.

THE GENETIC CODE TRIPLET CODE 3 bases in the DNA code for one amino acid in the protein. Each triplet is known as a CODON. UNIVERSAL Found in all organisms. DEGENERATE More than one codon for each amino acid. NON-OVERLAPPING START AND STOP CODONS

PROTEIN SYNTHESIS TRANSCRIPTION AMINO ACID ACTIVATION TRANSLATION

Protein Synthesis Summary

TRANSCRIPTION

U C A C U U G U A C A G G A A U U A G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A C U U G U A C A G G A A U U A G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A C U U G U A C A G G A A U A U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A C U U G U A C A G A A U A U G G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A U U G U A C A G A A U A U G C G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A U U G U A A G A A U A U G C C G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A U U G U A A A A U A U G C C G G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A U U G U A A A U A U G C C G A G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A U G U A A A U A U G C C G A U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A U G A A A U A U G C C G A U U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A U G A A A U A U G C C G A U U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A G A A A U A U G C C G A U U G U T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U C A G A A U A U G C C G A U U G U A T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

C A G A A U A U G C C G A U U G U A U T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

C A A A U A U G C C G A U U G U A U G T A C G G C T A A C A T A C A A T C G U HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

C A A A U A U G C C G A U U G U A U G U T A C G G C T A A C A T A C A A T C G HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

C A A A A U G C C G A U U G U A U G U U T A C G G C T A A C A T A C A A T C G HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

C A A A U G C C G A U U G U A U G U U A T A C G G C T A A C A T A C A A T C G HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

C A A U A G C T A C G G C T A A C A T A C A A T C HELICASE unwinds and unzips the relevant part of the DNA helix. RNA POLYMERASE attaches to the DNA. One DNA strand acts as the template (SENSE STRAND), the other is redundant (ANTISENSE STRAND). As RNA POLYMERASE moves along the sense strand, ribonucleotides are assembled in a precise order due to complementary base pairing (A <-> T/U ; G <-> C).

U A G C Fully formed mRNA peels off the DNA and leaves the nucleus via a nuclear pore. The DNA rewinds.

U A G C Fully formed mRNA peels off the DNA and leaves the nucleus via a nuclear pore. The DNA rewinds.

U A G C

U A G C

AMINO ACID ACTIVATION

THE ANTICODON DETERMINES WHICH SPECIFIC AMINO ACID IS ATTACHED ACTIVATION OCCURS WHEN THE tRNA COMBINES WITH A SPECIFIC AMINO ACID U A C THE ANTICODON DETERMINES WHICH SPECIFIC AMINO ACID IS ATTACHED

TRANSLATION

A ribosome binds to the mRNA near the START CODON. U U A C G C U A A C A U G C C G A U U G U A U G U U A G A ribosome binds to the mRNA near the START CODON.

C A U U A G G C U A C A C A U G C C G A U U G U A U G U U A G tRNA with the complementary ANTICODON (UAC) binds to the start codon (AUG) held in place by the large subunit of the ribosome. It brings with it the amino acid methione.

The ribosome now slides along the mRNA to “read” the next codon. U U A G G C U A C A C A U G C C G A U U G U A U G U U A G The ribosome now slides along the mRNA to “read” the next codon.

C A U U A U A C A C G G C A U G C C G A U U G U A U G U U A G A second tRNA now bind to this codon, bringing a second amino acid with it.

A peptide bond is formed between the two amino acids. U U A U A C A C G G C A U G C C G A U U G U A U G U U A G A peptide bond is formed between the two amino acids.

C A U U A C U A A C G G C A U G C C G A U U G U A U G U U A G The tRNA which carried the first amino acid is released but leaves its amino acid behind as a DIPEPTIDE.

The ribosome now slides along the mRNA to “read” the next codon. U U A C U A A C G G C A U G C C G A U U G U A U G U U A G The ribosome now slides along the mRNA to “read” the next codon.

One by one each codon is read as the ribosome moves along the mRNA. U U A C U A A C G G C A U G C C G A U U G U A U G U U A G One by one each codon is read as the ribosome moves along the mRNA.

C A U U A C U A A C G G C A U G C C G A U U G U A U G U U A G Each time the growing polypeptide is linked to the amino acid on the incoming tRNA.

C A U U A C G C A A C U A U G C C G A U U G U A U G U U A G

U A C G C A C A U A C U A U G C C G A U U G U A U G U U A G

U A C G C A C A U A C U A U G C C G A U U G U A U G U U A G

U A C U A G C C A U A C A U G C C G A U U G U A U G U U A G

U A C U A G C C A U A C A U G C C G A U U G U A U G U U A G

U A C U A G C C A U A C A U G C C G A U U G U A U G U U A G

U A C U A U A C G C A C A U G C C G A U U G U A U G U U A G

The polypeptide is complete when the ribosome reaches the STOP codon. U A C U A U A C G C A C STOP ! A U G C C G A U U G U A U G U U A G The polypeptide is complete when the ribosome reaches the STOP codon.

The polypeptide is released. C U A C C A U U A G C A U G C C G A U U G U A U G U U A G The polypeptide is released.

The polypeptide is released. C U A C C A U U A G C A U G C C G A U U G U A U G U U A G The polypeptide is released.

The polypeptide may combine with other polypeptides and will become variously coiled/folded to produce a protein.

G C U A The tRNAs are recycled.

A U G C C G A U U G U A U G U U A G The mRNA may be used again in this form, or it may be broken down into nucleotides which can be reassembled to produce a different polypeptide.

The ribosome is free to move along another mRNA.

Ribosomes work in groups so that many slide along a mRNA molecule simultaneously. These groups are called POLYRIBOSOMES. Each ribosome takes about 1 minute to travel along a mRNA molecule.

QUIZ What sort of chemical is helicase? Why is DNA double stranded if one strand is redundant? Where in the cell are the ribosomes? What is the start codon? Give the three alternative stop codons. Give the primary structure (sequence of amino acids) of the polypeptide made in this animation. What is the difference between a polypeptide and a protein? What is the advantage of ribosomes operating as polyribosomes? What are the similarities and differences between DNA replication and protein synthesis?

ANSWERS Helicase is an enzyme and therefore also a protein. DNA is double stranded to permit replication. Ribosomes are located in the cytoplasm. The start codon is AUG. The three stop codons are UGA, UAG and UAA. Give the primary structure of the polypeptide is methionine, proline, isoleucine, valine, cysteine. Polypeptides have less than 100 amino acids, protein have more. A protein may consist of several polypeptides. Polysomes increase efficiency, they enable one mRNA molecule to produce many polypetides simultaneously. HINT - Think about the enzymes and nucleotides used, the end product and the location of the process.

Really good website http://www.le.ac.uk/ge/genie/vgec/he/index.html

What is a genome? All of the genetic material (the base pairs) found in one complete set of an organism’s chromosomes. The study of genomes is called genomics.

Does genome size matter? COMMON NAME SPECIES NAME Approx GENOME SIZE (millions of base pairs) Fruit fly Drosophila melanogaster 180 Snake Boa constrictor 2100 Human Homo sapiens 3100 Onion Allium cepa 18000 Lungfish Protopterus aethiopicus 140000 Amoeba Amoeba dubia 670000 Why would a single celled animal like the amoeba need a genome that is about 200 times larger than the human genome? Ans: They carry a lot of junk DNA!

What is a gene? Segment of DNA that codes for formation of a protein Structural genes express structural and/or functional proteins. Regulatory genes are short nucleotide sequences that express proteins that control the activity of structural genes by feedback mechanisms.

Number of genes COMMON NAME SPECIES NAME No. GENES Human Homo sapiens 25000 Mustard plant Arabidopsis thaliana 27000 Fruit fly Drosophila melanogaster 14000 Baker’s yeast Saccharomyces cerevisiae 6000 Gut bacterium Escherichia coli 4000 Should we be offended that a mustard plant has as many genes as a human?

An Overview of Gene Structure Coding Region DNA sequence that will be transcribed from the template strand. 5’ 3’ 5’ STOP Regulatory region START 3’ Promoter region Terminator region

Gene Expression The expression of genetic information is one of the fundamental activities of all cells. Instructions stored in DNA are transcribed and translated into various RNA molecules.

Introns and Exons The coding region in eukaryotes contain: introns - non-coding regions of DNA exons - coding regions of DNA Prokaryotes do not have introns – why? They don’t carry “junk DNA” due to short replication cycles

RNA Processing in Eukaryotic Cells DNA Template Strand Pre-mRNA transcript of DNA template strand Introns are spliced out by spliceosomes leaving only the sequences that will be expressed. This is an example of RNA processing. The introns usually are degraded. The result is a mature mRNA strand that will leave the nucleus to be translated. INTRON INTRON EXON EXON EXON Spliceosome Spliceosome EXON EXON EXON

Genome to proteome The human genome has about 25,000 genes but our proteome (the total number of different proteins) is much larger (~100,000) How can this occur? Many genes can produce more than one protein because the mRNA transcript contains different combinations of exons. This process is called alternative splicing.

Alternative splicing INTRONS Pre-mRNA transcript Possible mRNAs using different combinations of exons Result: when each mRNA is translated, a different protein is produced. EXON 1 EXON 2 EXON 3 EXON 4 EXON 1 EXON 4 PROTEIN 1 EXON 2 EXON 3 EXON 2 EXON 1 EXON 4 PROTEIN 2 EXON 2 EXON 3 EXON 4 PROTEIN 3

Gene Regulation Each cell contains an entire organism’s genome. All cells of an organism have the same genome, but can have different phenotypes. For example, cells in your eye have the gene for producing fingernail protein (keratin) but this gene is not expressed. How do genes get switched on or switched off?

Why regulate gene expression? Cells conserve energy and materials by blocking unneeded gene expression. If a substrate is absent in the environment why produce the enzyme for that substrate! Repressor molecules keep the cell from wasting energy by not transcribing mRNA or making enzyme molecules that have no use. The cell can control its metabolism – resources are used only when there is a metabolic need and can be redirected to other metabolic pathways.

Gene regulation in prokaryotes Bacteria have groups of genes that are controlled together and are turned on/off as required. E.g. the lac operon is a set of genes in bacteria used for lactose metabolism. Bacteria produce the enzymes to break down lactose to glucose and galactose only when lactose is present. http://www.sumanasinc.com/webcontent/animations/content/lacoperon.html http://pages.csam.montclair.edu/~smalley/LacOperon.mov

Lac Operon – an example of gene regulation in E. Coli The bacterium Escherichia coli is capable of producing the enzyme b-galactosidase which splits lactose to produce glucose and galactose. This enzyme is only produced when the bacteria encounters lactose. When lactose is not present, a protein binds to the promoter region of the b-galactosidase gene and prevents transcription (RNA polymerase cannot access the promoter). This protein is referred to as a repressor protein. When lactose is present in the growth medium of the bacteria, it enters the cell and binds to the repressor protein causing it to be removed from the DNA and allowing transcription to occur. The gene is ‘on’ or ‘off’ depending on the nutrients available to the cell.

Gene regulation in Eukaryotes Still being investigated Proteins involved (like prokaryotes) – but more complicated Enhancers: act as binding sites for activator proteins and increase number of DNA polymerase molecules transcribing genes Chemical modification: eg presence or absence of histone proteins

Other factors in gene regulation It is important to realize that the environment of a cell can also influence the expression of genes. This includes factors such as: Light Temperature Ions Hormones