Unit 2 - Gene Interactions

Nucleosome

Repeating subunit of chromatin in eukaryotic cells

Heterochromatin

DNA tightly bound to nucleosome

F Plasmid

Has genes that assist with transfer of that plasmid to another host bacterial cell

R Plasmid

Has genes that confer antibiotic resistance

Lateral or Horizontal Gene Transfer

Transfer of genetic material between bacteria, archaea, other organisms

Conjugation

Transfer of replicated DNA from donor to recipient through pilus, single stranded plasmid is replicated as it leaves/enters

Transformation

Uptake DNA from environment

Transduction

Transfer of host DNA via a virus accidentally incorporating host genome

tra_

Genes that help transfer F plasmid

oriT

Origin of transfer

traA

Structural subunit of F pilus

Conjugation of F plasmid

1. F⁺ assembles pilus

2. Relaxosomes binds at oriT to cleave the T strand of DNA

3. Relaxosomes partially degrade, leaving relatase bound at 5’

4. Exporter move relaxase-T complex & recipient and replication begins

High Frequency Recombination

Bacterium with F plasmid integrated into main chromosome

Conversion of F to Hfr

Insertion sequence lines up and integrates into a giant chomosome

Hfr with F^-

Donor bacterial chromosomal genes and maybe partial F plasmid genes are transferred to the recipient because pilus degrades

Transformation Steps

1. dsDNA enters at receptor where a strand is degraded

2. Transforming strand pairs with homologous region

3. Cell division makes a transformant and 1 nontransformant

Competent

Cell capable of being transformed

Transductant

Donor DNA integrated into recipient’s chromosome forms

Prophage

Bacteriophage integrated into a bacterial host chromosome

Genetic Complementation Analysis

Analyzing mutated bacteriophages with plaques to determine if it’s on the same or different genes

Even Plaques from Genetic Complementation Analysis

Mutation is on different genes that the bacteriophages complement each other

No Plaques from Genetic Complementation Analysis

Mutations are on the same genes, so no complementation

Abnormal Plaques from Genetic Complementation Analysis

Homologous recombination causes a copy of the gene to be normal

Major Grooves in DNA allows

Base pairs to be exposed from larger spacing

Conversative Replication

Synthesized DNA has no parental DNA

Semiconservative Replication

Synthesized strand has half parental and new

Dispersive Replication

Synthesizes strand has sections of parental

Messelson-Stahl Experiment

1. Replicated DNA in N¹⁵

2. Allowed 1 cycle of replication in N¹⁴ twice

3. Ultracentrifuged tubes and observed where the DNA suspended

Replisome

Complex of proteins at replication fork

Helicase

Unwinds double helix

DNA Topoisomerase

Relaxes supercoiling

SSB

Prevents strands from resealing

Primase

Synthesizes DNA primers

DNA Polymerase 3

Synthesizes DNA

DNA Polymerase 1

Removes and replaces RNA primers with DNA

DNA Ligase

Joins DNA segments

DNA Replication

1. Helicase breaks DNA and topoisomerase binds

2. SSB binds and primase synthesizes RNA primers

3. DNA poly 3 makes new strand

4. DNA poly 1 removes and replaces RNA priemrs

5. DNA ligase joins okazaki fragments

Base Pair Mismatch

DNA strand swings down and 3’ to 5’ exonuclease cleaves off the base pairs at the 3’ end

Telomeres

Repetitive sequences that ensures important information isn’t lost

Chromatin Memory

Turns off genes are immediately remembered to be turned off once replicated finishes

Telomere Synthesis

1. Telomerase attaches to empty strand where primers were removed

2. RNA template grows leading strand in a repetitive sequence

3. DNA poly extends other strand

4. Hanging strand knots upon itself

Ribonucleoprotein

Enzymatic complex that has protein and ribonucleic acid

Hayflict Limit

Number of times somatic cells can divide, around 50-70

PCR

Uses temperatures to denature DNA, DNA primers and Taq polymerase replicates DNA

Thermus Aquaticus

Bacteria that tolerates high temperatures and has Taq polymerase

PCR Ingredients

• DNA template

• dNTPs

• Taq polymerase

• 2 DNA primers

• Buffer

Sanger Sequencing

Determines nucleotide sequence by separately using a single type of ddNA and combining them together

Dideoxynucleotide

Lacks 3’ OH, which stops DNA synthesis

Sanger Sequencing Integredients

• DNA strand

• DNA primers

• DNA polymerase

• DNA & dDNA

Capillary Electrophoresis

Uses a tube and a camera to capture fluorescently tagged dDNA as they cross a detector

Coding Strand

Non synthesized strands that’s identical to RNA made

mRNA

Encodes sequence of amino acids may be polycistronic in bacteria and archaea

rRNA

Helps large and small subunits form

small nuclear RNA

In eukaryotic nuclei where many snRNA joins with proteins to make spliceosomes

microRNA

Eukaryotic regulatory RNAs that base pair with mRNAs, changing stability and efficiency of translation

small interfering RNA

Eukaryotic regulatory RNA made from long ds molecules that are cut into smaller pieces to regulated stability and translation

Telomerase RNA

In telomerase, ribonucleoproteins complex that acts a template to maintain and elongate telomere length of chromosome

Consensus Sequence

Most common sequence (-35 and -10)

Pribnow Box

-10 consensus sequence AT rich in prokaryotes

RNA Poly Holoenzyme

Core RNAp and specific signma subunit

Sigma Subunit

Guides RNA poly to promoters, and different versions changes binding specificity

Synthesis is in the

5’ to 3’

Hairpin Loop

RNA folds back on itself from GC rich areas binding strongly, followed by weaker AU regions forcing polymerase off

Intrinsic Bacterial Transcription Termination

Hairpin loop forms

Rho-Dependent Bacterial Transcription Termination

Hairpin + ATP active protein forces unbinding

Euchromatin

Lightly packed, open chromatin for gene expression

RNA Polymerase 1

Several ribosomal RNA genes

RNA Polymerase 2

Protein coding genes and most small nuclear RNA genes

RNA Polymerase 3

tRNA genes, small nuclear RNA genes, ribosomal RNA gene

Saturation Mutagenesis

Individually mutate every base pair and areas where transcriptions decrease, it’s a promotoer

Band Shift Assay

Combine a section of DNA with a promotor binder, if a promotor binds, it’ll be slower in a gel and prolly a promotor

DNA footprint Protection Assay

DNA is incorporated with DNase that’ll randomly cut DNA but promotor bound with be protected causing a gap in gel

Enhancer Sequence

Increases level of transcription of specific genes by bringing certain gene promotors close to RNA polymerase to bind

Activator Protein

Binds to enhancer to bring complex to RNA polymerase II

5’ Capping

Methylated guanine added to 5’ end of the first 20-30 mRNA made to stop degradation and increases translation efficiency

3’ Polyadenylation

Poly A tail replaces some mRA at the 3’ to end transcription

Group I and II Introns

Self splicing, chloroplast, mitochondria related

Pre mRNA Introns

Requires splicesomes

5’ Splice Site

Beginning of intron with GU nucleotides

3’ Splice Site

End of introns with AG nucleotides

Branch Site

Conserved sequence near the end of introns connecting 5’ intron end to branch point A within branch site recognition sequence

Branch Point Adenine

A nucleotide within branch site

Polypyrimidine Tract

Sequence of pyrimindines near the end of to promote spliceosome aseembly

Intron Recognition and Cleavage

1. snRUP U1 binds to 5’ and U2 binds to branch site

2. snRUP U4, U5, U6 binds forming inactivate splicesome

3. U4 dissociates, activating complex to cleave 5’ end

4. 2’ to 5’ bond form with branch point A stabilizing lariat intron

5. 3’ splice creates a free 3’ OH on exon 1, freeing the lariat

Alternative Promoters

Different transcription start points are different for cell types

Alternative Polyadenylation

Different ending sites

Alternative pre-mRNA Splicing

• Constitutive splicing

• Exon skipping

• Intron retention

• Mutually exclusion exons

Constitutive Splicing

All introns removed

Exon Skipping

Some exons are skipped

Intron Retention

Some introns retained for regulation

Mutually Exclusive Exons

Different exons are combined

Calcitonin

Derived from CALCA with exon 4 retained

CGRP

Derived from CALCA without exon 4

Species with introns tends to have

Small variable breeding population (N_e), causing weak natural selection from stronger genetic drift

rRNA and tRNA are

Transcribed, but not translated

E. Coli

7 pre RNA genes, each with the same rRNA genes but different tRNA all in 1 transcript

rRNA and tRNA in Eukaryotes

From different pre RNA transcripts

ETS

External transcribed spacer

ITS

Internal transcribed spacer

tRNA Processing

1. 5’ and 3’ ends trimmed

2. tRNA folds into 3D structure

3. Addition of bases