Lecture 01, Eukaryotic Gene Structure & Organization PDF

Genome, Transcriptome and Proteome ❖ DNA is the repository of genetic information (blueprint of life) that every cell in human body has the same DNA. ❖ Genome is the sum of all genetic material of an organism that provides an overview of the complete set of genetic instructions provided by th...

Genome, Transcriptome and Proteome ❖ DNA is the repository of genetic information (blueprint of life) that every cell in human body has the same DNA. ❖ Genome is the sum of all genetic material of an organism that provides an overview of the complete set of genetic instructions provided by the DNA. ❖ Transcriptome is the sum of all transcripts (mRNA) that investigates gene expression patterns of a cell. ❖ Proteome is the sum of all the proteins (dynamic protein products and their interactions) of a cell. 5 BIOC 405 – Lecture 1  Metabolome ❖ Metabolome is the qualitative and the quantitative collection of all low-molecular-weight molecules (metabolites) present in the cell that are participants in general metabolic reactions and that are required for the maintenance, growth, and normal function of a cell. ❖ The metabolome is a result of the biochemical reactions being catalyzed by the proteins of the proteome. This in turn determines the biological structure and function of the final phenotype of the organism. Genes determine what may happen and metabolites define what has happened. 6 BIOC 405 – Lecture 1  Organization of Prokaryotic Genome ❖ In prokaryotes: DNA is found in two structures 1. Chromosomal DNA → single, circular molecule of DNA localized in the nucleoid area of cytoplasm and contains nearly all the cell’s genetic information. The DNA appears to be attached at one or more points to the inner surface of the plasma membrane. The nucleoid (meaning nucleus-like) → is an irregularly-shaped region within the cell of a prokaryote that contains all or most of the genetic material. In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a nuclear membrane. 2. Extrachromosomal DNA → in form of plasmids in cytoplasm Plasmids are present in bacteria, archaea and some eukaryotic 7 organisms as yeast cells. BIOC 405 – Lecture 1  Organization of Prokaryotic Genome Plasmid DNA a) Small circular double stranded (ds) DNA molecule that replicate independently of chromosomal DNA because plasmid carries information required for its own replication → So, Plasmids are considered replicons, units of DNA capable of replicating autonomously within a suitable host. b) Plasmids are usually 1-5% the size of bacterial chromosome ranging in size from a few thousands bp to a few million bp. c) It carries genes needed for organism’s survival as colicins (antimicrobial proteins or bacteriocins) and antibiotic resistance genes. d) Many bacterial cells contain one or more plasmids e) Plasmids can frequently be transmitted from one bacterium to another (even of another species) mostly through conjugation (Horizontal gene transfer (HGT)). f) plasmid isn’t considered as genome because it doesn’t contain all the genetic information required for the organism. 8 BIOC 405 – Lecture 1  Organization of Prokaryotic Genome Prokaryotic genome organization 1. Most genome is coding. 2. Small amount of non-coding sequences as promotors and operators. 3. Their haploid circular genomes (one copy of genetic material) → (0.5 – 10 Mbp, 500-104 genes) → usually Operon, where all genes that are involved in the same asexual reproduction. so, prokaryotic genomes are process are located on the same place and under the haploid. control of one promotor & transcription of operon gives polycistronic mRNA containing more than 4. Contain operons and polycistronic transcription units. messages, each is translated independently. 5. Contain environment-specific genes on plasmids (extrachromosomal DNA) and other types of mobile genetic elements (MGEs) or mobilome. 9 BIOC 405 – Lecture 1  Organization of Prokaryotic Genome 6. Transcription and translation take place in the same compartment (coupled processes) Prokaryotes have primitive nuclear structure (i.e., their nuclear material (DNA) isn’t enclosed by nuclear membrane) This affects the transcription and translation processes making these processes coupled i.e., occur in the same site (cytoplasm) and occur at the same time (coupled processes). so, mRNA transcripts are translated as soon as they are formed → there is no post- transcriptional modification occurs to the mRNA transcripts → this means that prokaryotic transcripts have no introns i.e., no splicing occurs 10 BIOC 405 – Lecture 1  Organization of Eukaryotic Genome ❖ In eukaryotes: they contain 1. Nuclear DNA → DNA is present in form of chromosomes in the nucleus 2. Extrachromosomal (extranuclear) DNA → DNA is present in mitochondria and chloroplasts (in plants and algae) ❖ Eukaryotes have real or true nucleus → their nuclear material (DNA) is enclosed by a nuclear membrane. so, there is a separation between the transcription and translation processes. 11 BIOC 405 – Lecture 1  Organization of Eukaryotic Genome Eukaryotic genome organization 1. Most genome is non-coding (98%) i.e., there is a limited number of genes compared to the whole genome size. 2. With introns (discontinuous coding regions). Introns → intervening non expressed sequences that interrupt the expressed regions (exons) in eukaryotic genes. 3. With regulatory sequences as introns, promotors, and enhancers. 4. With repetitive DNA sequences. 5. Eukaryotic genome is organized as chromosomes (multiple pieces of linear DNA). 6. With multiple genome → nuclear and organellar genomes → organelle genome in energy house as mitochondria and plastids (chloroplasts) → their genome is circular ds DNA and resembles prokaryotic genome. 12 BIOC 405 – Lecture 1  Organization of Eukaryotic Genome 7. Monocistronic transcription units (mRNA molecules carry only one message) 8. Transcription and translation take place in different compartment 9. Often diploid genomes and obligatory sexual reproduction 10. Standard mechanisms of recombination during meiosis (crossing over). 13 BIOC 405 – Lecture 1  Organelle Genome ❖ Genes located on extranuclear chromosome (organelle genome) codes for about 5% of the RNA and polypeptides required for the organelle’s replication and functions while nuclear DNA codes for the remaining 95%. ❖ Organelle genomes are incomplete genomes because these organelles depend on some nuclear genes for their functions (not complete autonomous) ❖ Limited autonomy of mitochondrial (mt) genomes 14 BIOC 405 – Lecture 1  Mitochondrial Ribosomes ❖ The prokaryotic 70S ribosome has a small 30S and a large 50S subunit. The 30S subunit consists of one 16S molecule of rRNA and about 21 proteins, while the 50S subunit consists of two rRNAs (5S and 23S) and 31 proteins. ❖ 16S ribosomal RNA (or 16S rRNA) is the RNA component of the 30S subunit of a prokaryotic ribosome. It binds to the Shine-Dalgarno sequence (SD) that serves as a ribosomal binding site in prokaryotic mRNA, generally located around 8 bases upstream of the start codon AUG and helps recruit the ribosome to the messenger RNA (mRNA) to initiate protein synthesis. ❖ 23S rRNA has an essential function of peptidyl transferase that plays role in the peptide bond formation. ❖ Ribosomal 5S RNA (5S rRNA) is an integral component of the large ribosomal subunit in all known organisms with the exception only of mitochondrial ribosomes of fungi and animals. It is thought to enhance protein synthesis by stabilization of a ribosome structure. 15 BIOC 405 – Lecture 1  Mitochondrial Ribosomes ❖ Mammalian mt-ribosomes have small 28S and large 39S subunits, together forming a 55S mt- ribosome. 16 BIOC 405 – Lecture 1  Human Mitochondrial Genome 1. Small (16.5 kb) circular DNA → 1 gene/0.45 kb 2. With 37 rRNA, tRNA and protein encoding genes a) 24 of 37 genes are RNA coding I) 22 mt tRNA II) 2 mt ribosomal RNA (23S, 16S) b) 13 of 37 genes are protein coding (synthesized on ribosomes inside mitochondria) 3. All rRNA components are expressed only from mt-genome because all mt-protein coding genes are synthesized on ribosomes inside the mitochondria and not translated in cytoplasm. 4. mt-genome has its own genetic code that differs from that used by the nuclear genome (i.e., genetic code is near universal). So, mitochondria have its own tRNA components ex) UGA (stop codon, nuclear) → trp (mitochondria) 17 BIOC 405 – Lecture 1  Mitochondrial tRNAs How do human mitochondria manage to translate mRNAs into proteins with only 22 tRNA species? ❖ Mitochondrial tRNAs are able to do an extreme form of wobble in which U in the anticodon can pair with any other of the four bases in the third codon position of mRNA, allowing four codons to be recognized by a single tRNA. ❖ An unusual mode of wobble base interaction with mRNA codons exists in mitochondria. Uridine at the wobble position base pairs with all four nucleotides, while modification from uridine to 5-carboxymethylaminomethyluridine (cmnm5U) prevents pairing with pyrimidines. 18 BIOC 405 – Lecture 1  Wobble Hypothesis ❖ In the genetic code, there are 43 = 64 possible codons (3 nucleotide sequences). For translation, each of these codons requires a tRNA molecule with an anticodon with which it can stably complement. If each tRNA molecule is paired with its complementary mRNA codon using canonical Watson-Crick base pairing, then 64 types of tRNA molecule would be required. ❖ In the standard genetic code, three of these 64 mRNA codons (UAA, UAG and UGA) are stop codons. These terminate translation by binding to release factors rather than tRNA molecules, so canonical pairing would require 61 species of tRNA. Since most organisms have fewer than 45 types of tRNA, some tRNA types can pair with multiple, synonymous codons, all of which encode the same amino acid. ❖ In 1966, Francis Crick proposed the Wobble Hypothesis to account for this. He postulated that the 5' base on the anticodon, which binds to the 3' base on the mRNA, was not as spatially confined as the other two bases and could, thus, have non-standard base pairing. ❖ Crick creatively named it for the small amount of "play" or wobble that occurs at this third codon position. Movement ("wobble") of the base in the 5' anticodon position is necessary for small conformational adjustments that affect the overall pairing geometry of anticodons of tRNA. 19 BIOC 405 – Lecture 1  Wobble Hypothesis ❖ As an example, yeast tRNAPhe has the anticodon 5'- GmAA-3' and can recognize the codons 5'-UUC-3' and 5'-UUU-3'. It is, therefore, possible for non-Watson– Crick base pairing to occur at the third codon position, i.e., the 3' nucleotide of the mRNA codon and the 5' nucleotide of the tRNA anticodon. ❖ Therefore, a wobble base pair is a pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules. The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine (inosine)-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). ❖ Wobble base pairs are fundamental in RNA secondary structure and are critical for the proper translation of the genetic code. 20 BIOC 405 – Lecture 1  Human Mitochondrial Genome (cont.) 5. With very few repeats 6. No introns → 93% coding 7. Genes are transcribed as multimeric transcripts 8. Maternal inheritance 9. There are overlapping genes in mt-DNA ex) Two overlapping genes encoded by same strand of mt DNA (ATPase 8/ ATPase 6) (unique example) → not found in nuclear genome → giving good investment using small genetic material to produce many proteins 21 BIOC 405 – Lecture 1  Overlapping Genes ❖ Overlapping genes (OLGs) also called “dual-coding genes”, are two adjacent DNA segments that are partially or entirely overlapped with each other through a shared genomic location. So, a nucleotide sequence may make a contribution to the function of one or more gene products. ❖ Overlapping genes are translated in two different reading frames to yield two different proteins. ❖ These genes are present in the viral, prokaryotic, and eukaryotic genomes. 22 BIOC 405 – Lecture 1  Human Nuclear Genome 1. 3200 Mb → 30,000 genes → 1 gene/100kb 2. Introns in most of the genes 3. 1.5 % of DNA is coding 4. Genes are transcribed individually (no operon) 5. Repetitive DNA sequences (45%) 6. Recombination at least once for each chromosome 7. Mendelian inheritance occurs for X and autosomes (22 pairs) but not for paternal Y-chromosome 23 BIOC 405 – Lecture 1  Homologous Recombination ❖ Recombination occurs when two molecules of DNA exchange pieces of their genetic material with each other. ❖ One of the most notable examples of recombination takes place during meiosis (specifically, during prophase I), when homologous chromosomes line up in pairs and swap segments of DNA. This process, also known as crossing over, creates gametes that contain new combinations of genes, which helps maximize the genetic diversity of any offspring that result from the eventual union of two gametes during sexual reproduction. ❖ Genetic diversity occurs because certain physical characteristics, like eye color, are variable; this variability is the result of alternate DNA sequences that code for the same physical characteristic. ❖ These sequences are commonly referred to as alleles that are associated with a specific trait are only slightly different from one another, and they are always found at the same location (or locus) within an organism's DNA. 24 BIOC 405 – Lecture 1  Genomic Paradoxes ❖ There are three genomic paradoxes K-value is the chromosome number N-value is the gene number in the genome The haploid genome size of eukaryotes (Human 3200 Mb) 27 BIOC 405 – Lecture 1  N-value Paradox ❖ N-value paradox is due to potentiality of proteome and transcriptome diversity so that there is no need for increase in genes number. Solution 1 to the N-value paradox: Many protein-encoding genes produce more than one protein product (e.g., by alternative splicing or by RNA editing) → mechanisms that increase the protein coding capacity of the genome Solution 2 to the N-value paradox: We are counting the wrong things, we should count other genetic elements (e.g., small RNAs). Solution 3 to the N-value paradox: We should look at connectivity rather than at nodes. 28 BIOC 405 – Lecture 1 N-value Paradox 29 BIOC 405 – Lecture 1