MICB 211 Chapter 8 Notes

Genetic Organization

Phenotype

-physical features and functional traits of an organism

-reflection of proteins it possesses at any point in time

-proteins are grouped into functional categories

Genotype

-particular set of genes an organism possesses

-organism’s genotype resides in its genome

Gene

• unit of genetic information

• segment of DNA that possesses information specifying the order (= sequence) of amino acids in a polypeptide molecule

• sequence of nucleotides in a stable RNA molecule (tRNA or rRNA)

• not all genes specify polypeptides

• some genes specify rRNA or tRNA as their final gene product

Genome

• total physical DNA that specifies an organism’s characteristics → all of its genetic materia

• composed of DNA making up an organism’s gene and DNA between its genes

• genomics — study of genomes

• bacterial genomes constitute its chromosome and plasmids

Proteome

• all of the different proteins that can be synthesized by an organism

• proteomics — study and analysis of proteomes

Chromosome

• eukaryotes

  • chromosomal DNA is linear

  • organellar DNA is circular

• prokaryotes

  • genome components are circular DNA

  • DNA replication is initiated by proteins which bind at a specific nucleotide sequence — origin of replication oric

  • DNA synthesis proceeds in both directions (bidirectionally) around chromosome to a termination region opposite oriC on the chromosome

  • all prokaryotic genomes must have the same general classes of chromosomal genes

    • eg. genes encoding structural proteins, enzymes, transport proteins, DNA replication proteins, proteins for transcription and translation, regulatory DNA binding proteins, genes encoding tRNAs and rRNAs

  • different organisms have different mixes of genes within these classes (genotype) that endows them with particular characteristics and abilities (phenotype) linked to microenvironments they have adapted to

Plasmids

-bacterias contain genes outside their chromosome

-small circular pieces of DNA

-most plasmids not essential for viability of host under all environmental conditions but can augment a prokaryote’s ability to survive in certain circumstances

-often grouped according to genes they carry

• characteristics they give host organism in which they exist

-prokaryotic cells can host single or multiple copies of different plasmids at the same time

-acquired via horizontal gene transfer

• can be acquired by a cell without it needing to divide so genotype of an existing cell can change without it needing to reproduce

-theta bidirectional

• many plasmids are replicated like the chromosome

• beginning at origin of replication with specific nucleotide sequence (oriV) followed by bidirectional complementary DNA synthesis around dsDNA circle

Resistance Plasmids

• carry genes specifying proteins that confer resistance to antibiotics and other toxic chemicals

Nutrition Plasmids

• carry genes which specify enzymes that allow organisms to use certain molecules as nutrients

• molecules sometimes unusual chemicals (petroleum, pesticides) in the environment that are not essential for survival

Infection Plasmids

• carry genes which specify proteins that are not required for establishment of infections in animals and plants

Symbiosis Plasmids

• carry genes which specify proteins that are required for establishment of symbiotic relationship with plants and animals

Gene Structure

Individual Genes

-most prokaryotic genes are protein encoding

• specifies a polypeptide rather than a stable RNA

-typical bacteria gene consists of

• promoter region

  • RNA polymerase binds during transcription

• operator region

  • regulatory protein can bind

  • depending on type of regulation → promotes or inhibits transcription

• coding region

  • contains the template for mRNA

• terminator

  • RNA polymerase falls off the DNA to end transcription

Operons

-prokaryotic cells have evolved to have operons

-gene clusters

-multiple genes under the control of a single promoter

-common to have genes clustered together in the genome if they specific proteins that are involved in the same cellular processes

-genes of operons are transcribed together as a single mRNA initiating at one promoter but 2 or more different proteins with different amino acid sequences are translated from the single mRNA

Gene Expression

Transcription

Initiation

-transcription begins at a promoter, region of dsDNA about 50 bp in size

-promoters have specific nucleotide sequences that RNA polymerase recognizes to initiate transcription

RNA Polymerase

• bacteria have a single RNA polymerase

• binds nonspecifically to DNA and slides along it scanning for promoter sequence

  • once detected, polymerase stops → transcription initiated → sigma subunit dissociates

• exists in 2 forms:

  • holoenzyme

  • core enzyme

    • 4 subunits

      • 2 alpha subunits

      • 2 beta subunits

    • must associate with a 5th protein (sigma factor) to form holoenzyme

-sigma subunit confers the ability to recognize a promoter sequence

• different sigma subunits have different sequence specificity for promoter recognition

• allows bacteria to regulate the transcription of entire categories of genes by controlling whether particular sigma factors are made

Elongation

-coding region acts as the template for mRNA

-after RNA polymerase binds to promoter → RNA polymerase separates DNA strands to gain access to nucleotides of template strand

-enzyme moves along DNA strand in 3’ to 5’ direction

• synthesizes mRNA in 5’ to 3’ direction

• uses nucleotide sequence of template strand as guide for choosing complementary nucleotides

-first nucleotide copied into mRNA (+1 site) occurs a few nucleotides downstream of promoter

Termination

-intrinsic termination

• RNA polymerase falls off template strand when it pauses over a stretch of weakly H-bonded A-U nucleotide pairs

• RNA polymerase pausing associated with formation of stem-loop structure in mRNA that interacts with enzyme

-intrinsic transcription terminator — stem-loop structure

Translation

Initiation

-nucleotide sequence of mRNA is used by ribosomes as a guide to link amino acids together in a particular sequence resulting in a unique polypeptide

-for translation to begin, ribosomes specifically bind to mRNA at a nucleotide sequence (ribosome binding site - rbs)

• varies somewhat in sequence depending on gene but all are ~5 nucleotides in length

Elongation

-after binding to mRNA at rbs

• with participation of tRNA → ribosomes move along mRNA in the 5’ to 3’ direction joining amino acids together via peptide linkages in the sequence specified by nucleotide sequence in mRNA

-several ribosomes can sequentially bind to and simultaneously translate the same mRNA

-translation can start even before mRNA synthesis is finished

-during translation, nucleotides are read by ribosomes and tRNA in groups of three (codons)

• codons specify the amino acids according to the genetic code

Termination

-polypeptides are synthesized from the N-terminus (first) to C-terminus (last) so 5’ end of mRNA coding region corresponds to N-terminus of polypeptide and 3’ end of mRNA coding region corresponds to the C-terminus

-when ribosome encounters stop codon (UAG, UAA, UGA) translation stops and polypeptide is complete

Regulating Gene Expression

-proteins are expensive to make

-formation of each peptide linkage consumes 5 ATP and proteins have hundreds of amino acids

-translation of a single protein requires thousands of ATP molecules

-controlling when, where and how much of a particular gene product is made

Constitutive (Housekeeping) Gene Expression

• some genes expressed at all times because product of gene is required for a basic cellular function required at all times

Developmentally Regulated Gene Expression

• some genes must be expressed at different phases of a process or growth so that different proteins are made at different times

Cell-Specific Gene Expression

• all cells that make up prokaryotic organism contain the same genetic information

• some genes are expressed only in certain cell types

Environmentally Regulated Gene Expression

• some genes are expressed only under certain environmental conditions

Promoter Strength

-frequency in which RNA polymerase initiates transcription

-strong promoters initiate transcription more frequently than weak promoters

• strong promoters = RNA polymerase spends more time bound to DNA = higher binding affinity = greater likelihood transcription is initiated = gene transcribed and encoded polypeptide synthesized

-for a particular sigma subunit → all promoters have similar nucleotide sequences to an extent

• different RNA polymerase binding characteristics

Regulatory/Transcriptional Proteins

-recruits or inhibits RNA polymerase by actively binding to operator region of promoter

Lac Operon Example

• lac operon has genes that express enzymes involved in lactose metabolism

• cell only expresses proteins when lactose is the substrate

  • wasteful for cell to express proteins when there is no lactose to metabolize

  • lactose molecules must help regulate when lac operon is expressed or not

  • LacI gene is upstream of lac operon

    • encodes for transcriptional regulator

    • transcriptional repressor → binds to operator region that’s downstream of promoter (site where RNA polymerase and sigma factor binds)

• Lactose absent

  • LacI bound to operator preventing expression of lac operon

• Lactose present

  • LacI regulator binds to lactose → changes conformation → LacI can’t bind to operator → RNA polymerase able to transcript lac operon genes → express lac operon enzymes

CAP Site Example

• upstream of lac promoter is CAP site

  • cAMP binds to CAP → changes conformation → able to bind to CAP site

  • CAP is an enhancer that recruits RNA polymerase

    • promotes expression of lac operon

• amount of glucose in environment inversely related to amount of cAMP produced in cell

• low glucose → cAMP and CAP binds to CAP site → RNA polymerase binds to promoter → high transcription

• high glucose → no cAMP → CAP can’t bind to CAP site → basal levels of transcription

Enhancer/Activator

-enhances transcription

Repressor

-inhibits transcription

mRNA Degradation

-mRNA is subject to enzymatic degradation by ribonucleic enzymes (RNAses) as soon as it’s made

-mRNA = unstable RNA

-different mRNAs have different half-lives in prokaryotic cells

-some have half lives of ~1 minute while others have a half-life ~1 hour

-longer mRNA lasts in cell → more polypeptide copies can be translated from it

-only a few copies of a polypeptide may be translated from a short-lived mRNA whereas 100s of copies of polypeptide may be translated from a long-lived mRNA

Protein Degradation

-most costly mean of regulating gene expression

-directly degrade proteins that cell doesn’t need

-costly because cell has already spent resources to express and produce protein

-proteins meant for degradation → tagged and sent to large protease complex to be recycled back into their peptide or amino acid forms

Horizontal Gene Transfer (HGT)

-organisms obtain genes from nonparental sources

-organism that donates DNA = donor

-organism that receives DNA = recipient

Transformation

-transport of naked donor DNA from environment into recipient

-bacteria dies → lyse → release DNA and other cytoplasmic contents into environment

-DNA breaks into 100s of linear pieces/fragments

• average fragment carried ~20-100 average genes

• fragments can be taken up by other recipient cells

-bacteria have to be in competent state to transfer DNA from the environment

• proteins for transport of dNA from environment are synthesized

-com proteins — proteins or transport of DNA

Transduction

-transfer of DNA from donor to recipient mediated by virus (bacteriophage)

• result in change in phenotype of recipient

-binding of phage particle to cell surface receptor initiates infection process

-phage DNA injected into host cell cytoplasm and remained of T4 phage particle remains on cell surface

-depending on conditions and type of phage → bacteriophage can undergo 2 lifecycles

1. lytic life cycle

   • phage forces host to express its own viral proteins allowing for virus particles to assemble inside the host

   • virus lyses and releases newly formed particles into environment

2. lysogenic life cycle

   • integrates its viral DNA into host chromosome and stays dormant until certain conditions trigger lytic life cycle

-when new phage particles are being packaged in host → there’s a chance that fragmented host (bacterial DNA) is packaged into phage head by mistake

• transduction can occur because phage containing accidental bacterial DNA can go and infect another bacterial cell and transfer that DNA to a new host

-transducing phage — phage particle containing only host DNA

Bacteriophage

• infectious particles of genetic material wrapped in protein coat

• tailed dsDNA phage particles possess

  • protein capsids (heads)

  • coiled bodies

    • injects DNA into bacterial host

Conjugation

-transfer of plasmid from one prokaryote to another via cell-to-cell contact

-plasmids are genetic elements that can infect host cells

• exist only inside prokaryotic cells

• don’t have an extracellular state

-~25% of plasmids can directly transfer themselves from one organism to another via cell-to-cell contact and during conjugation

-plasmid-containing organism (donor) and organism receiving plasmid (recipient)

-conjugative plasmid possess DNA transfer or tra genes that encode transfer (Tra) proteins required to mediate the conjugative transfer of plasmid DNA from one cell to another

-initial contact between donor and recipient is made with conjugation pili

-proteins that compose conjugation pili are specified by some of the tra genes of conjugative plasmid in donor

-Type IV pili

-F-pili

-P-pili

Fate of HGT DNA

Plasmids

-plasmids can be introduced into cell via conjugation or transformation

-plasmids are stable → independently replicating entities can work autonomously from chromosome

ssDNA Fragments

-degraded by nucleotides via nucleases specific for ssDNA normally present in bacterial cells

-sometimes ss donor DNA fragments escape degradation because they interact with RecA protein

• potential for donor DNA to become integrated into recipient chromosome

-depends on whether donor DNA and recipient chromosomal DNA share homology

-2 regions of DNA exhibit high degree of nucleotide sequence similarity → homologous

-if 2 molecules of DNA share a region of homology of at least a few hundred nucleotides

• 2 molecules can be joined together at region of homology by homologous recombination

  • mediated by RecA protein

  • endonuclease (nickase) and ligase activities are involved in the process

• allows DNA fragment to incorporate itself into chromosome and genes may be expressed to alter or affect host phenotype