Chapter 8 - Microbial Genetics

Gene expression

• genetic information is used within a cell to produce the proteins needed for the cell to function

Gene Recombination

Genetic recombination can be transferred between cells of the same generation.

DNA replication 

Genetic information can be transferred between generations of cells.

Genetics

• The study of genes, how they carry information, how information is expressed and how genes are replicated

Chromosomes

structures containing DNA that physically carry hereditary information, the chromosomes contain genes

Genes

segments of DNA that encode functional products, usually proteins

Genome 

all the genetic information in a cell 

Genetic Code

• A set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein

Central Dogma

• DNA → RNA → protein

Transcription

DNA → mRNA

Gene expression

• A gene is expressed when we have the protein product

• In microbes, most proteins are either enzymatic or structural

Transcription

• DNA → mRNA

• scribe → copy

• staying in the same language of nucleic acid

DNA

• deoxyribonucleic acid (DNA)

  • Hydrogen bonds between the bases

  • strands are complementary

  • uniform width

  • antiparallel

  • order of the nitrogen-containing bases forms the genetic instructions of the organism

RNA

• single stranded 

• many different types 

• 5-carbon ribose sugar

• extra OH group

• contains uracil instead of thymine 

Transcription - general

• synthesis of a complementary mRNA strand from a DNA template

• transcription begins when RNA polymerase binds to the promoter sequence on DNA

  • does not transcribed

• proceeds in the 5’-3’ direction; only 1 of the two DNA strands is transcribed

• transcription stops when it reaches the terminator sequence on DNA

3 stages of transcription

• Initiation: RNA polymerase binds to a promoter

• Elongation: Synthesis by adding complementary nucleotides

• Termination: RNA polymerase reaches the terminator

Transcription

• RNA polymerase bind to the promoter, and DNA unwinds at the beginning of a gene

• RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA

• the site of synthesis moves along DNA; DNA that has been transcribed rewinds

• Transcription reaches the terminator

• RNA and RNA polymerase are released and the DNA helix re-forms.

Transcription - Eukaryotic Specific

• transcription occurs in the nucleus whereas translation occurs in the cytoplasm

• Exons are regions of DNA that code for proteins

• Introns are regions of DNA that do not code for proteins 

• Spliceosome → a large RNA-protein complex that removes introns and splices together exons 

  • composed of small nuclear ribonucleoproteins (snRNA) and RNA

RNA processing

In the nucleus, a gene composed of exons and introns is transcribed to RNA by RNA polymerase

Processing involves snRNPs in the nucleus to remove the intron-derived RNA and splice together the exon-derived RNA into mRNA

After further modification, the mature mRNA travels to the cytoplasm, where it directs protein synthesis.

Translation direction

• mRNA → protein → translate between two languages (nucleic acid to amino acid)

The components required for protein synthesis

• messenger RNA (mRNA): DNA → info → ribosomes

• Ribosome

  • ribosomal RNA (rRNA): integral part of ribosomes

  • Ribosomal proteins 

• Amino acids

  • Transfer RNA (tRNA): transports amino acids during protein synthesis

Translation

• mRNA is translated into the “language” of proteins 

• Codons are groups of 3 mRNA nucleotides that code for a particular amino acid 

• translation of mRNA begins at the start codon: AUG

• Translation ends at nonsense codons: UAA, UAG, and UGA

• Codons of mRNA are “read” sequentially

• tRNA molecules transport the required amino acids to the ribosome

• tRNA molecules also have an anticodon that base-pairs with the codon

• amino acids are joined by peptide bonds

• In bacteria, translation can begin before transcription is complete

The genetic code

• 61 sense codons encode the 20 amino acids

• The genetic code involves degeneracy, meaning each amino acids is coded by several codons

The regulation of bacterial gene expression 

• constitutive genes are expressed at a fixed rate

• other genes are expressed only as needed 

  • inducible genes

  • repressible genes

Pre-transcriptional control → Induction

• An inducible operon includes genes that are in the “off” mode with the repressor bound to the DNA, and is turned “on” by the environmental inducer. 

  • When turned “on”, induction turns on gene expression

  • initiated by an inducer, which binds to the repressor, turning it inactive

  • The default position of an inducible gene is off

Pre-transcriptional control - repression

• Repression inhibits gene expression and decreases enzyme synthesis

  • mediated by repressors, proteins that block transcription

• Repressible operon default: “on” mode → meaning the DNA gene is being expressed because the repressor is inactive

  • turned “off” by the environmental corepressor and repressor.

Operon Model of Gene Expression

• Promoter: segment of DNA where RNA polymerase initates (or promotes) transcription of structural genes

• Operator: segment of DNA that controls transcription of structural genes

• Operon: set of operator and promoter sites and the structural genes they control

• unique to prokaryotes 

• In an inducible operon, structural genes are not transcribed unless an inducer is present 

  • E.coli → enzymes of the lac operon are needed to metabolize lactose

  • In the absence of lactose, → repressor binds to the operator, preventing transcription

  • in the presence of lactose, the metabolite of lactose (allolactose → inducer) binds to the repressor

    • The repressor cannot bind to the operator, and transcription occurs

• In repressible operons, structural genes are transcribed until they are turned off

  • Excess tryptophan is a corepressor that binds and activates the repressor to bind to the operator, stopping tryptophan synthesis 

Lac operon - an inducible operon 

• Structure of the operon

  • promoter → operator → ZYA structural genes

  • operon is regulated by the product of the reg. gene (gene before promoter)

Lac operon → Repressor inactive

• I gene is transcribed and translated to make a repressor protein

  • transcription → makes repressor mRNA

• translation → makes active repressor protein

• The active repressor protein binds to the operator region of the operon

• When the repressor is bound to the operator → RNA polymerase can’t move forward to transcribe the structural genes (Y,Z, and A)

• as a result → transcription is blocked and the genes that normally make the enzymes 

Structure of the Lac Operon

• The lac operon controls the breakdown of lactose in E. coli 

  • includes 3 main region:

    • regulatory gene: makes the repressor protein, which blocks transcription 

    • control region: contains promoter and operator 

      • The promoter is where RNA polymerase binds to start transcription 

      • The operator is the “switch” that the repressor binds to, turning the operon off

    • Structural Genes (ZYA): code for enzymes that break down lactose 

      • lacZ: makes β-galactosidase (breaks lactose into glucose + galactose) 

      • lacY: makes permease (helps lactose enter the cell) 

      • lacA: makes transacetylase (detoxifies byproducts) 

• When the inducer (allolactose) binds to the repressor protein, the inactivated repressor can no longer block transcription. The structural genes are transcribed, ultimately leading to the production of the enzymes required for lactose catabolism. 

Trp operon - A Repressible Operon 

• The operon consists of the promoter and operator and structural genes that code for the protein 

• the operon is regulated by the product of the regulatory gene

• The Trp operon controls the production of enzymes needed to make the amino acid tryptophan 

• The regulatory gene → repressor mRNA → inactive repressor protein (cannot bind to the operator region) → allows RNA polymerase to attach to the promoter and trancribe the structural genes → resulting mRNA is then translated into enzymes that synthesize tryptophan

• the cell produces tryptophan when its levels are low 

When tryptophan levels are high:

• the amino acids acts as a corepressor

• tryptophan binds to the inactive repressor protein → changing its shape and activating it

• the active repressor then binds to the operator region of the DNA

  • blocks RNA polymerase from binding to the promoter or moving forward → transcription stops

  • no mRNA or enzymes are made

Summary: Repressor active → operon off.

The operon shuts down when enough tryptophan is present — a negative feedback loop that prevents waste of energy and resources.