BCHM Exam 3

• Alternative splicing

  Different splicing patterns from the same primary RNA transcript can produce different mRNA
• RNA editing

  Different mRNA can be produced from the same primary RNA transcript
• Regulating transcription in bacteria

  1. Use of σ factors

  2. Binding other proteins (transcription factors) to promoters
• σ factors


  Recognize different classes of promoters, allows coordinated expression of different sets of genes
• Transcription factors


  Bind to promoters of specific genes, may bind small signaling molecules to turn genes on/off, protein's affinity toward DNA is altered by ligand binding or posttranslational modifications, allows expression of specific genes in response to signals in the environment
• Specificity factor


  Provides specificity for DNA binding proteins that regulate gene expression, increases binding affinity for regulatory proteins rather than to a random DNA sequence
• Repressors


  Reduce RNA Pol-promoter interactions or block the polymerase, bind to operator sequences on DNA near promoter in bacteria, when repressor binds it blocks pol so transcription does not occur
• Effectors


  Can bind to repressor and induce a conformational change, change may increase or decrease repressor's affinity for the operator and thus may increase or decrease transcription
• Negative regulation


  Involves repressors, despite how repressor can have opposite effects on transcription, both are negative regulation ex. lac operon
• Positive regulation


  Involves activators, enhance activity of RNA polymerase when bound near promoters, activator may remain bound until a molecule signals dissociation, or may only bind when signaled ex. binding of CAP in lac operon
• Lac operon


  1. Genes are transcribed together, so mRNAs are several genes represented on one mRNA (polycistronic)

  2. LacI encodes for lac repressor, repressor always binds to o1, sometimes o2/3 forming a loop so RNA pol cannot bind to promoter, stopping lac operon gene transcription

  3. Allolactose can bind to repressor, allowing it to leave and for RNA pol to access promoter

  4. When glucose and lac repressor are absent - lac operon transcription Is stimulated by CRP-cAMP, cAMP binds to site near the promoter and stimulates transcription 50-fold

  5. Whether glucose is high or low, if lactose is absent, the repressor stays bound, no transcription even when CRP-cAMP bind

  6. When lactose is present, transcription depends on glucose level, repressor dissociates, but transcription is only stimulated significantly if cAMP rises
• Combinatorial control


  Transcription factors mix and match with different combinations to regulate different genes, allows more genes to be regulated with the same TF
• Transcriptional attenuation


  Transcription begins but is then halted by a stop signal (attenuator), relies on the fact that in bacteria, transcription and translation can proceed simultaneously, the attenuator sequence is in the 5'-region of a leader sequence, and it can make the ribosome stall
• Regulation of the trp operon

  1. Trp repressor binds to the operator when tryptophan is high, blocking the promoter so the enzymes are not transcribed

  2. When tryptophan is low, ribosome stalls at trp codons, RNA pol does not pause, segments 2 and 3 base-pair, transcription proceeds and the trp synthetic enzymes are made, no attenuation - transcription continues
• Regulation of the SOS response

  1. Normally, SOS genes are repressed by LexA repressor, LexA binds to operators at several genes

  2. Damaged DNA produces a lot of single strands, ssDNA is bound by RecA, activating its ability to interact with LexA repressor

  3. RecA binds to LexA repressor, causing it to self-cleave and dissociate from DNA, now all genes LexA repressed can be expressed
• Translational feedback for ribosome proteins

  Each operon for an r-protein encodes a translational repressor, if rRNA not around, repressor binds to mRNA and blocks translation (making ribosome), repressor has greater affinity for rRNA than for mRNA, so translation is repressed only when synthesis of r-proteins exceeds a level needed to make ribosomes
• Stringent response


  Lack of amino acids produces uncharged tRNA, uncharged tRNA binds to ribosomal A site, rRNA synthesis triggers a cascade that begins with binding stringent factor protein (RelA) to ribosome, stringent factor catalyzes formation of guanosine tetraphosphate (ppGpp), binding of ppGpp to RNA polymerase inhibits it, reducing rRNA synthesis & further ribosome synthesis
• Riboswitches


  Domains of an mRNA that can bind a small-molecule ligand, the binding of ligand affects conformation of the mRNA and its activity, allowing mRNA to participate in their own regulation and respond to changing concentrations of the ligand
• How riboswitches work

  1. Binding of ligand affects conformation of the mRNA and its activity

  2. Allows mRNA to participate in their own regulation and respond to changing concentrations of the ligand
• Basic steps of cloning

  1. Isolate a specific gene from the source organism and amplify it in the target organism

  2. Cut the source DNA at the boundaries of the gene with restriction endonuclease

  3. Select a suitable carrier DNA (vector)

  4. Insert the gene into the vector using DNA ligase

  5. Insert the recombinant vector into host cell via electroporation

  6. Let the host produce multiple copies of recombinant DNA
• Restriction endonucleases


  • Cleave DNA phosphodiester bonds at specific sequences
  • Palindromic sequences
  • Common in bacteria - eliminates infectious viral DNA
  • Some make staggered cuts (sticky ends)
  • Some make straight cuts (blunt ends)
  • Large number are known - commercially available
• DNA ligases


  • Enzyme that covalently joins two DNA fragments
  • Seals nick/gap in phosphodiester backbone
  • Normally function in DNA repair
  • Human DNA ligase uses ATP, bacterial DNA ligase uses NAD
• Plasmids

  • Can carry genes that give a host bacterium a resistance against antibiotics
  • Allows growth (selection) of bacteria that have taken up the plasmid
• Cloning vectors

  • Plasmids - circular DNA molecules that are separate from the bacterial genomic DNA
  • Can replicate autonomously - origins of replication for use in bacteria and/or yeast
  • Carry antibiotic resistance genes
  • Allows cloning of DNA up to 15,000 bp
• Expression vectors


  • Special plasmids that contain sequences that allow transcription of the inserted gene
  • Differ from cloning vectors by having promoter sequences, operator sequences, code for ribosome-binding site, transcription termination sequences
• Bacterial artificial chromosome (BAC)


  • The process for cloning via BAC is very similar to using plasmids
  • Often, a colorimetric approach using X-gal is employed to select for colonies containing the chromosomal insert
  • F par genes ensure only one or two plasmids are generated for each replication and that they are evenly distributed between the daughter vectors
  • Lac z gene disrupted when foreign DNA inserted, turns colonies white
• Yeast artificial chromosome (YAC)


  • Yeast are the simplest eukaryote for DNA recombination
  • They have linear chromosomes with telomere ends, which can be unstable in vitro
  • YAC is circular to accommodate quick replication and stable storage but has a removable segment that linearizes the product for transformation
  • Can restrict growth media with an essential AA - YAC would provide AA - only the YAC w/ DNA in it would grow
  • Used to clone whole chromosomes (up to 300,000 bp) - makes sure only a couple copies of the plasmid at most is in each cell & each daughter cell only gets one or two copies to reduce recombination
• Steps of PCR

  1. Mix together target DNA, primers (oligonucleotides complementary to target), nucleotides: dATP, dCTP, dGTP, dTTP, thermostable DNA polymerase

  2. Melt DNA at about 95°C

  3. Cool separated strands to about 50–60°C

  4. Primers anneal to the target

  5. Polymerase extends primers in the 5'->3' direction

  6. After a round of elongation is done, repeat the steps
• Taq DNA polymerase

  Used because it is an extremophile that can handle high temperatures
• Applications of PCR


  • Cloning - use flanking primers to replicate and amplify a specific segment of DNA
  • Site-directed mutagenesis (oligonucleotide-directed) - use "sandwich" primers to make small changes to the segment of DNA and then amplify the altered DNA
  • DNA fingerprinting
• Reverse transcriptase PCR (RT-PCR)

  Used to convert RNA code to DNA and amplify specific segments
• Quantitative PCR (qPCR)

  • Used to show quantitative differences in gene levels
  • How much are those genes actually being expressed under those conditions
  • Use fluorophores as probe, when in stable hairpin structure, fluorophore and quenching molecule quench each other so there will be no fluorescence. Probe prefers to linearize and bp with target DNA, causing fluorescence
• Green fluorescent protein (GFP)

  • Used to determine where protein is in vivo
  • Use recombinant DNA technologies to attach GFP to protein of interest - can visualize with a fluorescent microscope
  • Immunofluorescence - tag protein with primary antibody and detect secondary antibody containing fluorescent tag
  • Protein can also be fused to a short epitope, and the primary antibody detecting the epitope can be fluorescently labeled
• Site-directed mutagenesis


  1. Understanding the function of proteins often requires that a specific amino acid residue be mutated

  2. To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene

  3. Site-directed mutagenesis usually relies on chemically synthesized mutated primers that are incorporated into newly synthesized DNA

  4. Mutated plasmids are always sequenced to confirm that the desired (and only the desired) mutation is present

  5. Used to understand how a protein functions/ if an AA is critical for it to function

  6. A fragment of a recombination vector is cleaved with a restriction endonuclease and a synthetic DNA segment containing a mutation is inserted
• Construction of cDNA


  1. Eukaryotic genes have exons - coding regions and introns - noncoding regions

  2. Bacteria cannot splice introns out of coding DNA

  3. mRNA is intron-free genetic material, as the codons have already been spliced out

  4. mRNA can be extracted from eukaryotic cells

  5. All mRNA molecules have a poly(A) tail - helps in purification of mRNA/ serves as a universal template

  6. A cDNA strand can be synthesized using mRNA as a template

  7. The hybrid can be converted to double stranded DNA, known as cDNA - tells us the actual genes being expressed in a cell in their final form, more stable than mRNA, no introns
• cDNA libraries

  • Get all of the mRNA in a particular organism, use reverse transcriptase and put that on a chip and see what genes/what forms are being expressed at that moment
  • GFP tagged library allows us to see where protein goes
• Recombinant protein tags

  • Recombinant proteins can be tagged for purification because purification is difficult
  • The tag binds to the affinity resin, binding the protein of interest to a purification column
  • This washes away any impurities in the protein, once this has been done the protein can be removed from the column and it should be pure
  • May make protein fold better
• Coimmunoprecipitation

  1. Protein-protein interactions

  2. Protein complex isolation - one protein in the complex is epitope tagged

  3. Gentle isolation of epitope-tagged protein will also isolate stably interacting proteins

  4. All proteins isolated can be separated and identified

  5. Use of TAP tags has enhanced procedure - allows two purification steps, eliminating loosely associated proteins and minimizing nonspecific binding
• Yeast 2-hybrid screens


  1. The protein of interest is tagged with the GAL4-activation domain

  2. The DNA library with all yeast genes is tagged with Gal4-binding domain

  3. The reporter gene under the control of Gal4

  4. The differentially tagged proteins must interact in order to get expression of the reporter gene

  5. Yeast cannot survive unless the reporter gene is expressed

  6. Use cDNA library express it with activation domain to test to see if it will interact with your bait
• Microarray chips

  • Contain fragments from genes in the group to be analyzed - allow us to see differences in gene expression
  • Full genome of bacteria or yeast, or protein encoding families from larger genomes
  • mRNA or cDNA from different samples are differentially tagged
  • The microarray can be probed with mRNAs or cDNAs from a particular cell type or cell culture to identify the genes being expressed in those cells
  • Analysis on the same chip shows differences
  • DNA microarrays allow simultaneous screening of many thousands of genes; high throughput screening
  • Can be used for genome wide genotyping, tissue specific gene expression, mutational analysis