Introduction to Genetics, DNA Replication, and Protein Synthesis PDF

Introduction to Genetics Dr. Angela Parry-Hanson Kunadu 1 Learning objectives  Define heredity, genetics, genome, genes  Describe the structure and function of DNA  Describe the synthesis of DNA in bacterial cells  Apply the use of the Codon...

Introduction to Genetics Dr. Angela Parry-Hanson Kunadu 1 Learning objectives  Define heredity, genetics, genome, genes  Describe the structure and function of DNA  Describe the synthesis of DNA in bacterial cells  Apply the use of the Codon table  Explain the principles and mechanisms of protein synthesis or gene expression  Explain regulation of gene expression 2 The Cell  Nucleic material, contains DNA The general location and forms of genome in Eukaryotes, Procaryotes and Viruses 3 Introduction  Genetics: the science of genes, genetic variation and heredity in living organisms  Genome is sum total of genetic material (DNA) carried within a cell  Made up of all the genetic material in the cell including the chromosome and plasmids and non-chromosomal sites such as mitochondria and plasmids  Heredity: passing of traits from parents to their offspring through sexual or asexual reproduction  Chromosomes are cellular structures containing neatly packaged DNA that physically carry heredity information  Chromosomes contain genes  Genes are segments of DNA that code for functional products  Some viruses do not have DNA, they only posses RNA. In that case, genes are segments on RNA that code for functional products 4 Nucleic acid  Nucleic acids are genetic materials that serve as carriers of genetic information  There are two types: 1. Deoxyribose nucleic acid (DNA)  Storage of all genetic information necessary for cell functions 2. Ribonucleic acid  Copied information from DNA (genes) that are useful for expression of genes 5 DNA  Deoxyribose Nucleic Acid  It is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses.  It is the hereditary material of cells and considered the blueprint of life  Contains instructions (code) for construction of RNA and proteins  Function  Long term storage of information  DNA segments carrying genetic information are called GENES  Other DNA sequences have structural purposes or are involved in regulating use of genes 6 DNA  DNA is organized into long structures called CHROMOSOMES  During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell its own complete set of chromosomes  The DNA molecule may be circular or linear, and can be composed of 100,000 to over 3,750,000,000 nucleotides in a long chain  Generally, eukaryotic cells (cells with nuclei) have large linear chromosomes and prokaryotic cells (cells without defined nuclei) have smaller circular chromosomes. There are exceptions to this rule  Parts of the chromosome  1.Chromatid; 2. Centromere 3. Sort arm; 4. Long arm  3 and 4 are also telomeres 7 DNA topography  The circular chromosome is packaged by the action of a special enzyme called a topoisomerase  First, the linear DNA molecule is wound twice around the histone proteins, creating a chain of nucleosomes.  The nucleosomes fold in a spiral formation upon one another.  The spiral arrangement further twists on its radius to forma bigger supercoil 8  DNA properties  DNA molecules are made up of repeating nucleotide monomers  DNA contains 2 long polymers of repeating nucleotide units  Fundamental components of nucleotides  5 carbon sugar = RIBOSE (RNA) or DEOXYRIBOSE (DNA) sugar  Phosphate  4 different compounds containing nitrogen (nucleobases or bases)  The backbone of DNA is made up of sugars and phosphate group joined by ester bonds  These two strands run in opposite directions to each other and are therefore anti-parallel. 9 DNA properties  The DNA chain is 22 to 26 Ångströms wide (2.2 to 2.6 nanometres)  One nucleotide unit is 3.3 Å (0.33 nm) long  Twin helical strands form the DNA backbone  As the strands are not directly opposite each other, the grooves are unequally sized  The major groove, is 22 Å wide  The minor groove, is 12 Å wide 10 Simplified structure of nucleobase  The basic unit of DNA structure is a nucleotide, composed of phosphate, deoxyribose sugar, and a nitrogen base  Several units of a nucleotide makes up nucleic acids 11 Chemical structure 12 Structural difference between Deoxyribose and ribose sugar 13 Nucleobases: Purines pair with Pyrimidines Purines Pyrimidines Thymine 14 DNA  The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings  These asymmetric bonds mean a strand of DNA has a direction (5’ to 3’)  In a double helix the direction of the nucleotides in one strand is opposite to the direction in the other strand: the strands are “antiparallel”  The asymmetric ends of DNA strands are called the 5′ (''five prime'') and 3′ (''three prime'') ends, with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl group 15 Chemical structure of DNA: Nucleotide  Nucleotides consist of nucleobases, (deoxy)ribose and a phosphate group Ester bond Glycosidic bond Backbone 16  5’ has a phosphate end  3’ has a hydroxyl end 5’ 3’ direction Phosphodiester bonds between 3rd and 5th C 17 DNA structure  In a DNA double helix, each type of nucleobase on one strand normally interacts with just one type of nucleobase on the other strand  Complementary Base Pairing  Purines form HYDROGEN BONDS with pyrimidines, with Adenine bonding only to Thymine, and Cytosine bonding only to Guanine  The arrangement of two nucleotides binding together across the double helix is called a base pair 18  Adenine binds with Thymine A-T = 2 H bonds Guanine binds with Cytosine G-C = 3 H bonds Hydrogen bonds break and form easily 19 20 Summary: Structure of the DNA 21 DNA Replication 22 Flow of genetic information 23 Cell division  When a cell divides, it must replicate the DNA in its genome so that the two daughter cells have the same genetic information as their parent  Mechanism of DNA replication involves:  Separation of the double stranded DNA (unzipping)  Each strand's complementary DNA sequence is recreated by an enzyme called DNA polymerase  This enzyme makes the complementary strand by finding the correct base through complementary base pairing, and bonding it onto the original strand  As DNA polymerases can only extend a DNA strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix 24  DNA replication: 25 DNA Replication  DNA replication is initiated at particular points in the DNA, known as ORIGIN (ori) which are targeted by proteins that separate the two strands and initiate DNA synthesis.  Origins contain DNA sequences recognized by replication initiator proteins  These initiators recruit other proteins to separate the strands and initiate replication forks 26 Semi conservative DNA replication 27 Growth of the DNA strand  To start replication, the DNA polymerase enzyme needs a primer with available 3’-OH  New DNA strand grows in the 5’ to 3’ direction ONLY  The 2 phosphates at the end of incoming nucleotide are removed as pyrophosphate provides energy for the bond between the two nucleotides 28 Enzymes involved in DNA replication  DNA Gyrase relaxes supercoiling ahead of the replication fork  Topoisomerase is the enzyme that relieves tension to the DNA molecule by nicking and cutting certain places on the phosphate backbone  Helicase is the next enzyme that is involved in "unzipping" the DNA to produce two single strands of DNA  RNA polymerase, called primase, lays down the RNA primer  DNA polymerase III uses the RNA primer to start laying down new nucleotides on the single strand of DNA  DNA polymerase I replaces the RNA primer with DNA  DNA ligase joins the backbone of the newly formed DNA strands 29 DNA replication Synthesizes Okazaki fragments 30 Replication in E. coli 31 DNA replication  DNA is copied by DNA polymerase  In the 5 → 3 direction  Initiated by an RNA primer  Leading strand synthesized continuously  Lagging strand synthesized discontinuously  Okazaki fragments  RNA primers are removed and Okazaki fragments joined by DNA polymerase 1 and DNA ligase 32  DNA replication 33 Flow of genetic information 34  Application of the DNA code: Transcription and Translation 35 Learning outcomes  Explain the relationship between the structure of DNA and the structure of proteins  Describe the different structures of RNA and their basic functions in gene expression  Describe the genetic code, codons, and anticodons, and how they relate to one another  Explain the steps in transcription and translation  Describe the different types of control of gene expression 36 Summary of the flow of genetic information in cells  Although DNA is the blueprint of life, it cannot perform cellular processes.  The information in the genome has to be carried by RNA molecules in a process called transcription.  The information contained in the RNA is used to synthesize proteins. This process is called translation 37 The Gene-Protein Connection The Triplet Code and Its Relationship to Proteins  Gene sequences code for proteins.  Each protein is different therefore the information that translates into the protein also differs.  Within functional genes, information exist as triplets of codes called codons.  The differences in genes comes from the sequence arrangements of these codons  Each codon translates into one amino acid in a 3:1 ratio of nucleotides to amino acid  Proteins are made up of amino acids.  Proteins contribute significantly to the phenotype by functioning as enzymes and structural molecules. 38 Simplified view of the DNA-protein relationship  DNA is a blueprint that indicates which kinds of proteins to make and how to make them. This blueprint exists in the order of triplets along the DNA strands.  The order of triplets directs a protein’s primary structure— the order and type of amino acids in the chain—which determines its characteristic shape and function. 39 Ribonucleic acids (RNAs)  RNA is an encoded molecule like the DNA but its structure differs in a number of ways:  (1)It is a single-stranded molecule that can be folded into secondary and tertiary structures. The folding of RNAs determine the type and function of the RNA.  There are 3 types of RNA involved in transcription and translation:  Messenger RNA (mRNA)  Transfer RNA (tRNA)  Ribosomal RNA (rRNA)  (2) RNA contains uracil, instead of thymine, as the complementary base-pairing mate for adenine.  (3) Although RNA, like DNA, is structured with a backbone of alternating sugar and phosphate molecules, the sugar in RNA is ribose rather than deoxyribose 40  Messenger RNA, Transfer RNA & Ribosomal RNA Messenger RNA Transfer RNA Ribosomal RNA 41 Protein synthesis 42 Transcription  Transcription is the first stage of gene expression  Transcription involves synthesis of mRNA using codes on the DNA as a template.  Transcription proceeds in three stages:  Initiation  Elongation  Termination  Transcription begins when RNA polymerase binds to the promotor sequence (initiation). The promoter sequence consist of 2 sets of short DNA sequences before the initiation sequence.  Transcription proceeds in the 5 → 3 direction  Transcription stops when it reaches the terminator sequence  RNA synthesis has a proofreading mechanism that enables detection and removal of incorrectly added nucleotides 43  Transcription The function of the promoter is to provide a location for binding of the RNA polymerase. The promoter region is identified by a protein called the sigma factor. The sigma factor locates the promoter sequence and recruits the RNA polymerase onto the promoter sequence. The promoter regions are highly conserved regions. Here you can see the -10 TATA box, rich in Adenine and thiamine nucleobases. Also on this figure is the terminator region denoted by polyT. 44  Major events in transcription Each gene contains a specific promoter region and a leader sequence for guiding transcription initiation. DNA is unwound at the promoter by RNA polymerase. Only one strand of DNA, called the template strand, supplies the codes to be transcribed by RNA polymerase. This strand runs in the 3’ to 5’ direction. The RNA polymerase moves along the DNA template, adding nucleotides complementary to the template strand to elongate the mRNA. The mRNA strand reads in the 5’ to 3‘ direction. 45  Termination of transcription  The other strand—the non template strand—is sometimes called the coding strand because its sequence is the same order as the mRNA (although it will have thymine instead of uracil).  DNA that has already been transcribed rewinds back into its double helix structure.  At termination, the polymerase recognizes a site on DNA near the end of the gene that signals the separation and release of the completed mRNA. This is the termination site.  At the termination site, the mRNA transcript is released to be translated. 46 RNA synthesis  In eukaryotes, transcription is more complex because of the larger number of genes to be expressed, and the increased gene regulation required to control the expression of thousands of genes in hundreds of different types of cells.  This complexity is reflected in the three different RNA polymerase enzymes that transcribe different types of RNA molecules in eukaryotic cells.  RNAPI, RNAPII, RNAPIII  For example, RNAPI transcribes most rRNA genes whereas RNAPII transcribes most protein genes 47 48 Translation machinery  Translation is the process of decoding the information on mRNA into functional proteins  Translation requires the following tools:  Ribosomes  Transfer RNA  Messenger RNA  Amino acids  In prokaryotes, the mRNA molecule leaves the DNA transcription site and is transported directly to ribosomes. 49 Translation in Eukaryotes  In eukaryotes, transcription occurs in the nucleus mRNA processing in Eukaryotes  Translation occurs in the cytoplasm  Transcripts are synthesized as precursor mRNAs  Precursor mRNAs undergo major processing before export from the nucleus  A methylguanosine cap is added to the 5’ end of the precursor mRNA. This prevents the precursor RNA from being non-specifically degraded in the nucleus  The precursor RNA is spliced to remove the introns (interrupting genes) and join exons (contains expressed genetic information) together  A poly-A tail is added to the 3’ end of the RNA 50 Translation  mRNA is translated in codons (3 nucleotides)  Translation of mRNA begins at the start codon: AUG  Translation ends at a STOP codon: UAA, UAG, UGA  The codon is read on the mRNA  The complementary anti-codon is on the tRNA  tRNAs bearing amino acids are charged  Translation of mRNA occurs continuously 51  The Codon Table 52 Interpreting the DNA code  If the DNA sequence is known, the mRNA codon can be surmised.  If a codon is known, the anticodon and, finally, the amino acid sequence can be determined.  The reverse is not possible (determining the exact codon or anticodon from amino acid sequence) due to the redundancy of the code. 53  Stages in mRNA translation 54  Translation termination 55 Figure 8.10.7  Translation occurs continuously 56 Figure 8.11 Gene regulation: transcription  Although some ‘ housekeeping ’ genes are expressed for the life span of the organism, most genes are transcribed only for a specified period of time and then are turned off when the gene product is no longer needed  Genes can be regulated at several steps along the gene expression pathway  E.g. transcription start and stop signals for RNAP  Regulation signals are encoded on the DNA located upstream of the coding region. This is called the PROMOTER  RNAP and other proteins bind to the promoter to begin transcription 57 Promoters: Prokaryotes  The DNA sequence in the promoter region in prokaryotes is highly conserved  It includes a TATA box at -10 region (counted backwards from the 1st base transcribed into RNA) and another conserved sequence at -35  Prokaryotic genes also encode a sequence of bases that act as the transcription termination signal  In mRNA transcripts, the termination signal is a stem-loop structure that releases the mRNA from the DNA template 58 Promoters: Eukaryotes  Promoter sequences are much more varied, cover larger regions of the DNA and are located upstream of coding regions of genes  Some RNAPII promoters contain TATA sequences located 50bp upstream of transcription start site  Most protein coding genes of eukaryotes also contain additional control mechanisms called enhancers located large distances from the gene 59 Promoter: Eukaryotes 60 Control of gene expression: Prokaryotes  Many bacterial genes are organized into operons  OPERONS are small groups of related structural genes that are transcribed as a unit under the control of a promoter and an operator (an additional regulatory sequence upstream to the coding region; sometimes overlaps the promoter sequence)  Operons enable regulation of genes as a unit  Bacterial operons encode enzymes involved in biochemical pathways  In the E. coli genome, 75 different operons have been identified to control 250 genes  At any given time, repressor proteins bind operator sequences of genes to prevent transcription until needed  The operator is located near or overlaps the promoter sequence 65 Control of Gene expression: eukaryotes 66 67