FoB Unit4

What methods of gene expression regulation are used by prokaryotes?

• transcriptional

• post-transcriptional

• translational

• post-translational

What methods of gene expression regulation are used by eukaryotes?

• chromatin structure

• transcriptional

• post-transcriptional

• translational

• post-translational

Gene specific transcription factors (TFs)

• binds to DNA and regulates transcription

• activators (activate transcription)

• repressors (slow/stop transcription)

Negative control

regulatory proteins will slow/stop transcription

• repressors (transcription factor)

Positive control

regulatory proteins will increase rate of transcription

• activators (transcription factor)

Inducers

• molecule that binds to regulatory proteins and changes its shape and activity

found in BOTH eukaryotes and prokaryotes

Operator

• specific sequence of DNA near the promoter

ONLY in prokaryotes

What is the inducer for the lac operon?

lactose

How is the lac operon regulated?

inducible repressor

• without lactose, repressor blocks transcription

• with lactose, the inducer changes shape of repressor—> transcription occurs

How does low glucose levels but high lactose levels effect lac operon?

will produce the highest rates of transcription

• high cAMP inside the cell, lots of CAP activation

How does high glucose levels but low lactose levels effect lac operon?

will produce lowest rates of transcription

• low cAMP inside the cell, little CAP activation

CAP

transcription factor that increases the rate of transcription of lac operon

What part of the lac operon codes for the repressor?

lac1

How is lac operon transcription turned ON?

inducer binds to repressor and changes its shape—> can no longer bind to operator sequence

How is lac operon transcription turned OFF?

repressor is transcribed+translated from lac1 gene and binds to operator sequence—> blocks transcription

How do levels of lactose affect transcription levels of lac1?

Unaffected

• lac1 always encodes for repressor

How is the trp operon regulated?

repressible repressor

• with tryptophan, repressor binds to operator sequence—> no transcription

• without tryptophan, repressor is inactive—> RNA polymerase binds to promoter region

What’s the inducer for the trp operon?

tryptophan

Regulon

set of separate genes and operons that contain the same regulatory sequences

• are controlled by a single type of regulatory protein

Central Dogma of Biology

• DNA replication only occurs in S phase prior to mitosis or meiosis

• protein synthesis occurs almost all the time in almost all cells

Transcription

copying a DNA sequence into an RNA sequence

• carried out by RNA polymerase

Heterochromatin

tightly wound DNA due to histones

• transcription can’t occur

Euchromatin

less wound up DNA

• transcription can occur

General transcription factors (GTFs)

binds to promoter sequence

• the TATA box if present

• guides RNA polymerase

Introns

mRNA sequence that’s removed and stays INside nucleus

• removed before pre-mRNA becomes mature RNA

Exons

mRNA sequence that joins other exons and EXits the nucleus

Spliceosomes

removes introns from pre-mRNA and joins exons together

• composed of snRNP proteins

snRNP proteins

recognizes beginning and end of introns, indicating what sequences to remove

Alternative splicing

single pre-mRNA can be spliced in multiple different ways

• produces various mRNA molecules from a single pre-mRNA molecule

Methyl G cap

placed at 5’ end of mRNA molecule

• provides stability

Poly A tail

placed at 3’ end of mRNA molecue

• provides stability

Steps of transcription?

1) initiation

• chromatin shape, transcription factors, RNA polymerase

2) elongation

• DNA unwinds, RNA polymerase makes pre-mRNA

3) termination

• termination sequence, transcription stops

4) transcript processing

• alternative splicing

What is required for transcription to start?

GTFs must bind to promoter sequence

True or False: prokaryotes undergo alternative splicing

False

Promoter

• specific sequence of DNA located upstream

• starting point of transcription

found in BOTH eukaryotes and prokaryotes

Enhancer

• specific sequence of DNA

• binding site of activators (TFs)

ONLY in eukaryotes

Silencer

• specific sequence of DNA

• binding site of repressors

ONLY in eukaryotes

Somatic mutations

• occurs in body cells

• affects phenotype

NOT hereditary

Germ line mutations

• occurs in gametes

CAN be hereditary

Silent mutation

• changes 1 nucleotide

• amino acid stays same overall

Conservative missense mutation

• changes 1 nucleotide

• amino acid changes, but chemical property stays the same

Non-conservative missense mutation

• changes 1 nucleotide

• amino acid changes and chemical property changes

Nonsense mutation

amino acid changes to a stop codon

Frameshift mutation

• addition or deletion of a base

• significant effects on final protein

Impacts of mutations?

• impacts alternative splicing

  • spliceosome may not recognize where intron boundaries are

• impacts transcription factor binding

Do mutations impact DNA if it occurs before start codon?

no

Histones

• positively charged proteins that DNA winds around

• weaker charge found in euchromatin

• stronger charge found in heterochromatin

Nucleosomes

consists of 8 histones and DNA

Histone acetylation

• type of histone modification by adding acetyl group

• weakens positive charge, loosens DNA

Histone deacetylation

• type of histone modification by removing acetyl group

• strengthens positive charge, tightens DNA

DNA methylation

Restriction endonucleases

• cuts out gene and inserts into plasmid

• only cuts at recognition sites

Gene therapy

Approach to treating/curing disease via genome modification

Requirements for gene therapy

• Must be due to defects in a single gene

• Wild type allele sequence must be known

• Must be able to introduce therapeutic allele

Vectors

genetically-engineered viruses that contain therapeutic allele

In-vivo

Vector is injected into bloodstream

• in body

Ex-vivo

Cells are removed from patient, and then injected with vector

• out of body

CRISPR-Cas

• type of genome editing part of prokaryote immune system

• Store pieces of viral DNA in CRISPR locus known as spacers

Spacers

Short segments of viral DNA that bacteria incorporate into CRISPR region after infection

Cas9

• endonuclease that cuts DNA at specific sit

• Guided by crRNA+tracrRNA, or sgRNA

crRNA

Transcribed from CRISPR locus and acts as a guide for Cas9

• joins with tracrRNA

sgRNA

• Fusion of crRNA and tracrRNA

• Mixed with plasmid that contains gene for Cas9

Gene family

• group of genes that have similar DNA sequence

• Evolved from common ancestral gene via gene duplication

Nonhomologous end joining

• introduces deletions/insertions after Cas9 cuts gene

• Leads to disrupted gene function

Homology-directed repair

• homologous recombination

• Inserted by researchers

• Leads to corrected gene function

Polymerase chain reaction (PCR)

• amplifies specific segment of DNA

• 3 steps: denaturation, annealing, extension

Denaturation PCR step

• serves to break the hydrogen bonds of DNA

Annealing PCR step

• DNA primers bind to complementary DNA

• Consists of forward and reverse primers

Extension step

• Taq polymerase synthesizes new strands with dNTPS

Taq polymerase

heat-resistant enzyme for PCR

Symmetric cell division

produces 2 identical cells

Asymmetric cell division

produces 1 identical cell and 1 differentiating cell

Insulator

• regulatory gene sequence

• controls which genes enhancers activate