The Central Dogma DNARNAProtein DNA replication transcriptiontranslation

Prokaryotes : Relatively Simple….

Eukaryotes : Not so Simple….

Types of RNAs Produced in Cells Types of RNAs Functions mRNAsmessenger RNAs, code for proteins rRNAscomprise ribosomes tRNAsadaptors between mRNA and amino acids in protein synthesis snRNAssplicing of pre-mRNAs snoRNAsprocess and chemically modify rRNAs MicroRNAstranslation and mRNA degradation Other non-codingtelomere synthesis, X-chromo. RNAs inactivation, protein transport

Types of RNA in a Eukaryotic cell mRNA rRNA tRNA primary transcript, precursor to mRNA, rRNA, tRNA, before processing and cleavage. Contains introns which may serve as ribozymes snRNA (small nuclear) – plays structural and catalytic role in spliceosomes srpRNA – component of signal recognition particle (recognises signal peptides on proteins targeted to the ER) si (small interfering) RNA and micro RNA – involved in regulation of gene expression

DNA-binding proteins often fit into the major groove of the double helix

Model of RNA PolII Preinitiation Complex

Spatial Patterns of Gene Expression

Eukaryotic Gene Structure Three basic types of eukaryotic genes Pol I genes (rRNA) Pol II genes (protein coding genes, some small RNAs) Pol III genes (small RNAs such as tRNAs  Genes are the combination of a set of short exons with very variable lengths of introns,  The distance between genes is often much larger than the genes themselves. The problem of the function of introns, what is the role of the introns is the great challenge in molecular Biology.

Regulation of Gene Expression Most genes in any type of cell are not expressed Spatial – not every gene product needed in every cell type Temporal – Different genes expressed at different times –Environmental stimuli –Hormones (internal chemical signals)

Regulation of Gene Expression cntd. Level of expression of a gene usually refers to the amount of mRNA produced and rates of transcription Basic mechanism of regulation - similar in prokaryotes and eukaryotes: expression of genes is activated/inhibited through proteins interactions with DNA

GENE EXPRESSION IN EUKARYOTES: An EXPLANATION In Eukaryotes most genes have multiple overlapping regulatory mechanisms that operate at more than one level, from transcription through post translation (See figure). There are no operons in eukaryotes. However genes for related functions are often regulated coordinately.

Gene regulation in Eukaryotes Same principles as Prokaryotes, but additional levels of regulation: chromatin packaging DNA methylation (imprinting) mRNA processing mRNA transport out of nucleus transcription factors – proteins that bind to promoter region to enhance or prevent transcription gene silencing

Some promoter elements are required for transcription to begin. Eukaryotic protein coding genes contain both promoter elements and enhancer elements. Other promoter elements have a regulatory mechanism; these are specialized for the gene they control, binding specific regulatory proteins that control expression of the gene. Specific regulatory proteins bind also to the enhancer elements and activate transcription through their interaction with protein bound to the promoter elements.

Chromosome regions that are transcriptionally active have loose DNA- protein structures than chromosome regions that are transcriptionally inactive, resulting in sensitivity of the DNA to digestion by Dnase. The promoter regions of active genes typically have an even looser DNA-protein structure, resulting in hypersensitivity to DNase. Transcriptionally active genes exhibit lower levels of DNA methylation.

not all proteins are needed all the time in the cell Cells need to respond to changing environment by regulating cell processes Most cell functions are performed by enzymes/proteins Two levels of regulation: 1) amount of protein 2) activity level of protein Regulation is achieved in many different way

Regulation of the amount of a protein in the cell Rate of transcription mRNA stability/rate of degradation Rate of translation Protein stability/rate of degradation - Post-translational modification: glycosylation, phosphorylation, attachment of lipids/glycolipids -Binding to other proteins/substrate, etc

DNA in Eukaryotes is not Naked DNA complexed with equal mass of protein: chromatin Basic structural unit is nucleosome ~147 bp DNA wrapped around a protein core Protein core comprised of histones: (H2A, H2B, H3, H4) 2 Linker histone H1 joins nucleosomes together Histone tails subject to modifications that alter their highly basic charge Most common modification is acetylation of lysines

1400 nm 700 nm 300 nm Condensed chromosome Condensed chromatin Extended chromatin Nucleosomes DNA double helix Packed nucleosomes 2 nm Histone 11 nm DNA wound around a cluster of histone molecules 30 nm Scaffolding protein 30 nm fiber In Eukaryotes, DNA is packaged into chromosomes with the aid of proteins called histones (see CR Fig 19.2)

Model of the chromatin fiber from nucleosomes in a high order helix. The disklike nucleosomes associate to form a higher order helix with 6 to 8 nucleosome units per turn. The inter- actions stabilizing the fiber come from the histone H1, shown in red, thought to be at the center of the helix, and possibly also from the histone tail regions. [Adapted from F. Thoma, T. Koller and A. Klug, J. Cell Biol. 83: ] CHROMATIN STRUCTURE

Heterochromatin – highly condensed, non-expressed DNA Euchromatin – less condensed, accessible to transcription machinery, accessible to pancreatic DNase I

The inactive form of chromatin is the heterochromatin (arrow) and the active form is the euchromatin. In cells with a high level of activity, the euchromatin is increased. The nucleus in this cell contains a compact nucleolus (N). Nucleoli are often large, pleomorphic and multiple in neoplastic cells. Nucleoli are sites of rRNA synthesis and are rich in RNA.

Levels of Chromatin Packing Compaction - The length of the total DNA content of a human cell is nearly 1 meters. It must be packed into a nucleus about m in diameter. DNA packs in several bundles – chromosomes. The length of the DNA in each of human chromosomes is between 1.7 and 8.5 cm long. This is too long to fit into a cell. 

2. The chromosomal DNA is packaged into a compact structure with the help of specialized proteins called histones. The complex DNA plus histones in eucaryotic cells is called chromatin. 

3. The fundamental packing unit is known as a nucleosome. Each nucleosome is about 11nm in diameter. The DNA double helix wraps around a central core of eight histone protein molecules (an octamer) to form a single nucleosome. 

The Basis of Heredity All the information of cells is stored in their genetic material: DNA The entire complement of genetic material of an organism is called its genome. In most prokaryotes, the genome consists of one large molecule of DNA (usually circular), called a chromosome. A chromosome is a molecule made of DNA and proteins.

In eukaryotes, genes are NOT clustered in operons. Eukaryotic genes often contain non-coding introns ("intervening sequences") interspersed among the coding regions (exons). During RNA processing, introns are removed from RNA transcripts and the exons are spliced together.

Alternative RNA splicing in eukaryotes (not available in bacteria) different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns. H.A. Using this scheme explain the mechanism of Alternative RNA splicing.

Example of alternative splicing is the DSCAM gene in Drosophila. This single gene contains some 116 exons of which 17 are retained in the final mRNA. Some exons are always included; others are selected from an array. Theoretically this system is able to produce 38,016 different proteins. In fact, of 50 proteins synthesized from mRNAs, 49 of them turned out to be unique. Human : about 25 thousand genes, We probably make at least 10 times that number of different proteins. More than 50% of our genes produce pre-mRNAs that are alternatively-spliced.

.  Repeated regions. A large part of the human genome consists of repeated DNA. The (CCG)n and (CAG)n repeats expansion can cause several diseases in humans. 8 repeats 3 repeats The number of repeats is highly variable among individuals and the number of repeats is usually different on the two pairs of chromosomes of one individual). Analyzing the number of repeats provides a highly sensitive measure of individual identity and is the technique most often used for forensic DNA typing.

The Basis of Heredity Prokaryotes have haploid genomes (i.e. one copy of each gene, located on the single circle chromosome). Eukaryotes have diploid genomes (i.e. there are two copies of each gene which are located on pairs of chromosomes – one chromosome from each parental set) In eukaryotes, there are several chromosomes (always linear). 50 years ago new idea in GENETICS: Genes could be switch ON and OFF

A gene includes the entire functional unit: coding DNA sequences, non- coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. The estimate for the number of genes in humans are about 25,000 genes. H.A. Do you understand the words: “non-coding regulatory sequences” and “introns” ? Explain.

Chromosome: “Package" of genes and other DNA in the nucleus of a cell. Different kinds of organisms have different numbers of chromosomes. Humans have 23 pairs of chromosomes, 46 in all: 44 autosomes and two sex chromosomes. Chromosomes This figure schematically shows how DNA chains fold into a compact form in chromosome.

Chromosomes Bacterial chromosome - the DNA of a typical prokaryote like E. coli is contained in a single, large, supercoiled circular DNA molecule. Number of genes: between Eukaryotic chromosomes - The typical eukaryotic cell's genome is divided into several chromosomes, each of which contains a single, very large, linear DNA molecule (of 10 7 to 10 9 bp in length). Huge variability in genome sizes The number of eukaryotic chromosomes ranges from 1 (in an Australian ant) to 190 (in a species of butterfly).

regulatory sequence - a DNA sequence responsible for regulating gene expression, sites where regulatory proteins such as, for example, transcription factors bind preferentially. H.A. transcription factors?

Genome is defined as the sum of the genes of the haploid cell. A GENE one or more regions of a molecule DNA, which code for a particular protein. Bacterial gene Eukaryotic gene is a continuous region of DNA splits into separated segments in genomic DNA Exons (coding) segment Introns (not coding) Proteome - the entire collection of proteins that are encoded by the genome of an organism. H.A. What is the difference between the structures of Bacterial and Eukaryotic genes?

The genotype of an organism is the set of genes possessed by an individual organism. The phenotype is observable physical characteristics of an organism.