GENE 3200 Unit 4

purine (2 rings)

adenine and guanine

pyrimidine (1 ring)

cytosine, thymine, uracil

#of H bonds between A-T

2

#of H bonds between G-C

3

theta replication

• occurs in E coli and many other bacteria

• single origin (Ori) of replication

• single replication bubble and two replication forks

  • bidirectional replication from origin (whichever comes first)l

linear replication

• occurs in eukaryotes

• each chromosome has multiple origins

• each replication bubble has two replication forks

  • bidirectional replication from each origin

stages of DNA replication in E. coli

1. initiation: Ori

2. unwinding: SSBs, helicase, gyrase

3. elongation: primase, RNA primer, DNA pol III, leading/ lagging strands, DNA pol I, ligase

   1. termination: ligase

Initiation

• single replication origin (oriC)

  • initiator protein binds to oriC and causes a short section of DNA to unwind

Unwinding

• helicase binds to lagging strand, moves 5-3 and breaks H bonds

• SSBs stabilize single-stranded regions

  • DNA gyrase/ topoisomerase relieves torsional strain in front of each fork

elongation

• DNA synthesis is initated at RNA primers

• synthesized by primase, a RNA polymerase

  • RNA primers are complementary and antiparallel to each template strand

primase

synthesizes RNA primers that are complementary to OriC sequences

DNA poly III

synthesizes DNA in 5’-3’ direction

DNA poly I

has a 5’-3’ exonuclease that removes primer; polymerase replaces DNA. requires free 3’-OH from previous Okazaki fragment to begin function

ligase

seals nicks in DNA strands

polymerase chain reaction (PCR)

• generates billions of copies of one template DNA

  • takes only a few hours

oligonucleotides

short synthetic ss DNA that serve as primers for DNA synthesis in PCR.

right primer (reverse)

complementary to upper strand in PCR

left primer (forward)

complementary to lower strand in PCR

3 steps of PCR

1. denaturation

2. primer annealing

3. primer extension

Properties of DNA polymerases (Taq) for PCR

• optimal temp of >70C

• stable at high temps

• highly processive 5’-3’ polymerase

  • taq polymerase lacks 3’-5’ exonuclease activity

denaturation

sample heated at 90-100C to denature target DNA into single strands

primer annealing

temp reduced to 30-60C to allow hybridization of primers to their complementary sequence

primer extension

temp inc to 72C to allow DNA synthesis; extension time based of size of template

dideoxy nucleotide (ddNTP)

reaction stops when ddNTP is randomly incorporated because ddNTP lacks -OH group

gel electrophoresis

1. DNA samples containing fragments of diff sizes are placed in wells

2. an electrical current is passed through the gel

3. DNA fragments move to the positive pole, smaller=faster, larger=slower

Short Tandem Repeats (STRs aka Microsatellites)

short repeated DNA sequences; produce diff sized fragments when copied by PCR

STR profiling experiment

PCR and gel electrophoresis of a specific genetic locus

base substitution mutation

change one base to another; sequence length stays the same

transition mutation

base sub from A-G OR C-T

transversion mutation

base sub from purine-pyrimidine

insertion (indel)

addition of 1 nucleotide

deletion (indel)

subtraction of 1 nucleotide

promotor

determines where transcription initiates but not part of the transcribed sequence; where RNA poly binds to and transcription initiates

consensus sequence

the most common base or amino acid sequence found at a specific location on the promotor; can be strong or weak meaning transcription rate is high or low

DNA binding proteins

• can recognize specific sequences in DNA

• sequence can be very small

bacterial RNA polymerase

core RNA polymerase can’t bind to the promoter or initiate RNA synthesis without a sigma factor; alternative sigma factors give the holoenzyme specificity for diff promoter consensus sequences

steps of bacterial transcription

initiation, elongation, termination

bacterial transcription: initiation

• holoenzyme unwinds DNA

• sigma factor and core polymerase form the holoenzyme and bind to the promoter

• after initiation, sigma subunit disassociates from the core polymerase

sigma factors

• using different sigma factors allows coordinated expression of several genes

• recognize the promoter

bacterial transcription: elongation

• transcription bubble has 18 nt of unwound DNA

• topoisomerases relieve torsional stress

bacterial transcription: termination

Rho dependent or Rho independent

Rho dependent termination

• rho= ATP dependent helicase

• Rho recognizes and binds to Rut site in RNA: a C-rich region that lacks secondary structure

• Rho moves 5’-3’ along the RNA until in encounters RNA polymerase

• helicase activity of Rho causes pairing between DNA and RNA to destabilize leading to the release of RNA

Rho-independent termination

• consists of GC rich inverted repeats

• RNA poly pauses at the string of U’s allowing hairpins to form at the inverted repeats

• formation of the hairpin causes pairing between DNA and RNA to be destabilized leading to the release of RNA

genetic code qualities

• unambiguous: each codon specifies only one amino acid

• non-overlapping: the reading frame is set by the first codon used in translation

• nearly universal: same code used in nearly all organisms

• degenerate (redundant): most amino acids are encoded by more than one codon

tRNA

• an adaptor for translation of mRNA

• carries one specific amino acid

• each has a specific anticodon that is complementary to the codon for a specific amino acid

• tRNA and mRNA sequences are antiparallel

alignment of DNA, mRNA, and polypeptide

• amino group is at the N-terminus

• carboxyl group is at the C-terminus

E site of ribosome

Exit site: 5’ side of the codon, exit of uncharged tRNA

peptidyl (P) site of ribosome

site where new peptide bonds are formed between the growing polypeptide and the amino acid in the A site

aminoacyl (A) site:

3’ site of the codon, accepts charged tRNA

polyribose

mRNA in the process of being translated by multiple ribosomes

amino acids

have amino group, carboxyl group, and radical (R) group

lac Y

imports lactose into the cell

lac Z

metabolizes the lactose into monosaccharides

monocistronic DNA

one gene from one promotor, terminator, transcription and mRNA

polycistronic DNA

many genes under one promoter; organisms can group more than one gene to be transcribed together

operon

• a unit of DNA in which a group of genes is controlled by a DNA operator to which a repressor protein binds

• adjacent to a group of genes and would control their activity

• sensitive to a repressor produced by another gene; if present, the repressor inhibits transcription

• set of functionally related structural genes transcribed from a single promoter

structural genes

encode proteins that function in metabolism, biosynthesis, or structural aspects of the cell

regulatory genes

encode regulatory RNAs or proteins that control expression of structural genes and/or their products

• many transcriptional regulatory proteins have DNA-binding activity

• usually have its own p and t meaning its transcribed independently

regulatory elements

DNA sequences that are not translated into proteins but play a role in regulating other genes

• usually the binding site of regulatory proteins

operator

cis-acting regulatory sequence where trans-acting regulatory proteins bind and affect transcription

steps of lactose metabolism

1. permease actively transports lactose into the cell

2. where the enzyme B-galctosidase breaks into into galactose and glucose

3. B-galactosidase also converts lactose into the related compound allolactose

4. and coverts allolactose into galactose and glucose

lacI

• binds to the operator and prevents transcription of the lac operon in the absence of lactose

• lactose, when present, allosterically inhibits lacI binding to the operator, allowing transcription to occur

lac operon

• coordinated control of lactose metabolism

• lacI: repressor

• lacP: promoter

• lacO: operator

• 3 structural genes/proteins

  • lac Z= B-galactosidase

  • lacY= permease

  • lacA= transacetylase

• no lactose, lacI binds to lacO, lacZ/Y/A not expressed

• with lactose, lacI falls off lacO, lac Z/Y/A expressed

postitive regulation

proteins binding increases expression

negative regulation

protein binding decreases expression

repressible

signal present = decrease in expression

inducible

signal present = increase in expression

lacZ^-

nonfunctional b-galactosidase

lacY^-

nonfunctional permease

lacI^-

• defective repressor cannot bind to lacO

• constitutive mutation

• trans acting

lacI^s

• superrepressor, lacks allosteric site

• non-inducible

• trans acting

lacO^c

• fails to bind repressor protein

• constitutive

• cis acting

lacP^-

• defective promoter, no RNA polymerase

• non-inducible ?

• cis acting