Topic 9: Microbial Genetics

• Structure of a chromosome
  • Each human cell has about 1.8 meters of DNA
  • DNA is wound on histones → ~90 μm of chromatin
  • Chromatin is duplicated and condensed during mitosis
  • Results in ~120 μm of chromosomes
• Type of information stored in DNA molecule
  • Most chemical reactions dealing with organic molecules require enzymes
  • Enzymes are made primarily from protein
  • Information stored in DNA is simply how to place amino acids in a particular sequence to make proteins
  • Since the only structural variables within a DNA molecule are the nucleotides, the blueprint for producing protein must be contained within some type of code in the molecule
• Nucleic Acid Structure
  • DNA and RNA - nucleic acids
  • DNA and RNA are polymers of nucleotides linked together by phosphodiester bonds
  • Differences between DNA and RNA
  • nitrogenous bases they contain - T in DNA and U in RNA
  • sugars they contain - deoxyribose in DNA and ribose in RNA
  • single or double stranded - double in DNA and single in RNA
• Discovery of DNA Structure
  • James Watson, Francis Crick, Maurice Wilkins, and Rosalind Franklin
• DNA Structure
  • Adenine (A) + Thymine (T) pair by 2 hydrogen bonds
  • Guanine (G) + Cytosine (C) pair by 3 hydrogen bonds
  • Purines - adenine and guanine
  • Purimidines: thymine, cytosine
  • Double helix has major and minor grooves
  • Double helix has sugar, phosphate group, and nucelotide
• Eukaryotic DNA Organization
  • DNA highly organized in eukaryotic chromatin
  • Archaea DNA structure is similar to eukaryotic cells
  • form a ring or circle instead
  • Eubacteria use different proteins, not a histone but histone-like proteins
• The Bacterial DNA
  • Mostly single circular chromosome - haploid
  • Attached to plasma membrane (proposed)
  • DNA is supercoiled
  • ex: number of genes in E. coli = 3500-5000
  • Extra-chromosomal bacterial DNA: plasmids (1-5% of chromosome size)
• The organization of DNA in cells
  • In all archaea and most bacteria, DNA is circular double helix
  • Further twisting results in supercoiled DNA
  • Help organize the DNA into a coiled chromatin-like structure
• DNA Replication
  • Complex process involvng numerous proteins which help ensure accuracy
  • The two strands separate, each serving as a template for synthesis of a complementary strand
  • Synthesis is semi-conservation; each daughter cell obtains one old and one new strand
• The Replication Machinery
  • DNA polymerase catalyzes synthesis of complementary strand of DNA
  • DNA synthesis is in 5’ to 3’ direction resulting with the formation of a phosphodiester bond
  • Requires
  • A template - directs synthesis of complementary strand
  • A primer - DNA or RNA strand
• Strand Naming
  • (+) sense strand = coding strand = sense strand = non-template strand
  • (-) sense strand = non-coding strand = antisense strand = template strand
  • mRNA sequence = antisense strand
• Central dogma: DNA goes through transcription to become RNA which goes through translation to become protein
• Eukaryotic DNA Replication
  • Eukaryotic DNA is ~1,400 times longer than prokaryotic DNA and is linear
  • Many replication forks are used simultaneously
• Replication Enzymes
  • Helicase - separate DNA
  • Topoisomerase - regulate over underwinding of DNA (antibiotics, cancer target)
  • DNA gyrase - topoisomerase II, relieve strain (antibiotics target)
  • Primase - RNA polymerase, make a primer
  • DNA polymerase
  • DNA polymerase I - fill in primase/Okazaki fragement gaps
  • DNA polymerase II - back up
  • DNA polymerase III - primary, proof reading
  • DNA polymerase IV - prokaryotes involves in mutagensis
  • DNA polymerase V - prokaryotes SOS response due to damage
  • Replication direction 5’ →3’
• Prokaryotic DNA Replication
  • In most prokaryotes
  • bidirectional from a single origin of replication
  • some archaea have more than one origin
• Rolling Circle Replication
  • Some viruses and plasmids
  • Replicated by rolling-circle replication
• Establishment of the Genetic Code
  • Codon
  • genetic codw word (symbol)
  • composed of a three-nucelotide sequence
  • specifies an amino acid
  • Codon meanings deciphered by Marshall Nirenberg → resulted in Novel Prize in 1960s
• Gene Structure
  • Gene
  • Basic unit of genetic information
  • Also defined as the nucleic acid sequence that codes for a polypeptide, tRNA or rRNA
  • linear sequence of nucleotides with a fixed start point and end point
  • codons are found in mRNA and code for single amino acids
  • Reading Frame (ORF)
  • Organization of codons such that they can be read to give rise to a gene produce
• Genes
  • Eukaryotes
  • Genes are spread across multiple chromosomes, no operon, introns need to be spliced
  • Prokaryotes
  • genes are organized in operon, no introns
• Genes that Code for Proteins
  • Template strand directs RNA synthesis
  • Promotor is located at the start of the gene
  • is the recognition/binding site for RNA polymerase
  • functions to orient polymerase
  • among promoter sequence, TATA box for archaea and eukaryotes, Pribnow box for bacteria
  • Leader sequence is transcribed into mRNA but is not translated into amino acids
• Genes that Code for Proteins - The Coding Region
  • Begins with the DNA sequence 3’-TAC-5’
  • Produces RNA codon AUG
  • Codes for N-formyl methionine, a modified amino acid used to initiate protein synthesis in bacteria
  • coding region ends with a stop codon
    • immediately followed by a the trailer sequence which contains a terminator sequence used to stop transcription
• Chromosome Map of E. Coli
  • Chromosome length: ~1 mm
  • Cell Size: 0.5x3 μm
• The Flow of Genetic Information within a Single Cell
  • Process called gene expression
  • DNA divided into genes
  • Transcripiton yields a ribonucleic acid (RNA) copy of specific genes
  • Translation uses information in messenger RNA (mRNA) to synethesize a polypeptide
    • Also involves acivities of transfer RNA (tRNA) and ribosomal RNA (rRNA)
• Transcription in Eukaryotes
  • Eukaryotic genes
  • are split or interrupted
  • have exons (expressed sequences), regions coding for RNA that ends up in the mRNA
  • exons are separated from one another by introns (intervening sequences) that code for RNA that is never translated into protein
  • The initial RNA transcript has both intron and exon sequences - RNA processing required
  • Introns are removed from the initial RNA transcript by RNA splicing
  • splicing of the pre-RNA occurs in a large complex, the spliceosome, that contains the pre-mRNA
• Location of protein synthesis
  • Eukaryotic cells - DNA material located within the nucleus; protein synthesized outside of the nucelus
  • Prokaryotic cells - DNA and protein synthesis located in the cytoplasm
• Protein Synthesis
  • Anabolism or catabolism?
  • Anabolic reaction
  • Anabolism: “build up of large molecules”
  • Catabolism: “break down of large molecules”
  • Genetic code - universal (prokaryotes and eukaryotes) and degenerate (or redundant)
  • Transcription
  • produces 3 types of RNA
  • enzyme necessary
  • promoters and terminators
• The Ribosome
  • Eukaryotes
  • 80S ribsomes = 40S + 60S subunits
    • 40S has 18S subunit
    • 60S has 5s, 28S, and 5.8S subunits
  • mitochondrial and chloroplast ribosomes resemble prokaryotic ribosomes
  • Prokaryotes
  • 70S ribosomes = 30S + 50S subunits
    • 30S has 16S subunit
    • 50S has 5S and 23S subunits
• Structure of Ribosomes
  • composed of protein and rRNA
  • bacterial ribosomes are smaller than those of eukaryotic cells
• Ribonnucleic Acid (RNA) strcuture
  • Polymer of nucleotides
  • Contains the bases A, G, C, and U (uracil)
  • Sugar is ribose
  • Most RNA molecules are single stranded
• RNA production
  • An RNA molecule is produced by copying a short segment of a DNA molecule
  • The DNA molecule functions as a template as we saw for DNA duplication. However, only one side of the opened DNA molecule functions as a template
  • sense or antisense strand?
    • antisense → check answer
• Three Types of RNA
  • mRNA (messenger RNA) - a copy of a gene
  • DNA is the master blueprint for life that always remains within the nucleus. Since protein is synthesized outside of the nucelus, copies of genes on the DNA must be made for protein synthesis to occur in the cytoplasm.
  • rRNA (ribosomal RNA) - site for proetin synthesis
  • ribosomal RNA combines with protein to form ribosomes, which are the sites for proetin synthesis
  • attachement site: Shine-Dalgarno sequence (in arachaea and eubacteria)
  • tRNA (transfer RNA) - transports amino acids
  • transfer RNA molecules deliver amino acids to the ribosomes and place them in the appropriate location of the mRNA molecule
• RNA Processing
  • Gene (transcription into primary mRNA) → primary mRNA (removal of interons and joining of exons) → mature mRNA
  • No polyA tail, no CAP, or spliceosomes in prokaryotes (check)
• Genes That Code for tRNA and rRNA
  • tRNA genes have promotor, leader, coding, spacer, and trailer regions
  • leader, spacer, and trailer removed during maturation process → promoter & coding left
• Transfer RNA and Amino Acid Activation
  • attachment of amino acid to tRNA
  • catalyzed by aminoacyl-tRNA synthetases (tRNA ligase)
  • at least 20, need ATP
    • each specific for single amino acid and for all the tRNAs to which each may be properly attached (cognate tRNAs)
• Transcription
  • Initiation
  • Elongation
  • Termination
• Transcription Initiation
  • only a short segment of DNA is transcribed
  • promoter
  • site where RNA polymerase binds to initiate transcripition
  • is not transcribed
  • has specific sequence before transcription starting point
• Transcription Elongation
  • After binding, RNA polymerase unwinds the DNA
  • Prokaryotes - only 1 RNAP
  • Transcription bubble produced
  • Moves with the polymerase as it transcribes mRNA from template strand
  • Within the bubble a temporary RNA:DNA hybird is formed
• Transcription Termination
  • Occurs when core RNA polymerase dissociates from template DNA
  • DNA sequences mark the end of gene in the trailer and the terminator
• Transcription in the Archaea
  • RNA polymerase has similarities to both bacteria and eukaryotic enzyme
  • similarities with eukaryotes
  • archael gen promoters and binding of the RNA polymerase
  • introns present in some Archaeal genes
  • similarities with prokaryotes
  • mRNA is polygenic (=polycistronic)
• Transcription
  • RNA polymerase binds to the promotor sequence
  • proceeds in 5’ → 3’ direction
  • stops when it reaches terminator sequence
• Translation
  • produces the protein
  • sense codons vs. nonsense codons
  • anticodons - tRNA
  • Translation of mRNA begins at the start codon: AUG
  • RNA polymerase: Shine-Dalgarno sequence
  • Translation ends at a stop codon: UAA (ochre), UAG (amber), UGA (opal)
  • In prokaryotes - simultaneous transcription and translation → polyribsomes (polysomes): read multiple mRNA at the same time
• Organization of the Code
  • code degenceracy
  • up to six different codons can code for a single amino acid
  • sense codons
  • the 61 codons that specifiy amino acids
  • stop (nonsense) codons
  • the 3 codons used as translation termination signals
  • do not encode amino acids
• Translation
  • synthesis of polypeptide directed by sequence of nuelotides in mRNA
  • ribosome
  • site of translation
  • polyribosome - complex of mRNA with several ribosomes
• tRNA binding sites of ribosome
  • peptidyl (donor; P) site
  • binds initiator tRNA or tRNA attached to growing polypeptide (peptidyl-tRNA)
  • aminoacyl (acceptor; A) site
  • binds incoming aminoacyl-tRNA
  • exit (E) site
  • briefly binds empty tRNA before it leaves ribosome
• Wobble
  • loose base pairing
  • 3rd position of codon less important than 1st or 2nd
  • eliminates need for unique tRNA for each codon
• The Translation Process in Protein Syntheis (know picture)
• Coupled Transcripition and Translation in Prokaryotes
• Simultaneous Transcription and Translation in Prokaryotes (Polysomes)
  • Speed of expression
• Protein
  • domains
  • structurally independent regions of polypeptide
  • separated from each other by less structured portions of polypeptide
  • need ATP
  • native conformation - 3D tertiary structure
• Molecular chaperones
  • proteins that aid the folding of polypeptides
  • e.g. heat-shock protein
  • aid in transport of proteins across membranes
• Protein Splicing
  • removal of part of polypeptide before folding
  • inteins - removed portion
  • exteins - portions that remain in protein
• Final Phase in Elongation Cycle - Translocation
  • Three simultaneous events
  • peptidyl-tRNA moves from A site to P site
  • ribosome moves down one codon
  • empty tRNA leaves E site
• Termination of Protein Synthesis
  • takes place at any one of three codons
  • nonsense (stop) codons - UAA, UAG, and UGA
  • release factors (RFs)
  • aid in recogniton of stop codons
  • 3 RFs function in prokaryotes
  • only 2 RFs active in eukaryotes
• Summary
  • Location
  • Cytoplasm - prokaryotes
  • Nucleus - eukaryotes
  • Origin of Replication
  • 1 - prokaryotes
  • multiple - eukaryotes
  • Origin of Replication Size
  • 100-200 n - prokaryotes
  • 150 n - eukaryotes
  • Replication Bubble
  • 1 - prokaryotes
  • multiple - eukaryotes
  • Replication Fork
  • 2 - prokaryotes
  • multiple - eukaryotes
  • Okazaki Fragment Size
  • 1000-2000 n - prokaryotes
  • 100-200 n -eukaryotes
  • Speed per second
  • 2000 bp - prokaryotes
  • 100 bp - eukaryotes
  • Speed = Faster growth

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