Exam Three Flashcards

Frederick Griffith’s Experiments (1928)

• Demonstrated bacterial transformation

• Injected mice with different strands of bacteria

• Heat killed bacteria was transformed by living avirulant bacteria to become deadly to mice

Avery, McLeod, and McCarty Experiments (1944)

• Used test tube assays

• Experiments heavily implied that DNA is the transforming factor

Hershey and Chase Experiments (1952)

• Helped to confirm that DNA was the genetic material

• DNA and proteins of the phages were labelled to determine what was causing the infection

• Showed that when bacteriophages infect bacteria, their DNA enters the cell but most protein doesn’t

Nucleotides

• Building blocks of DNA

• Consist of:

  • A nitrogenous base

  • A pentose sugar

  • A phosphate group

Pyrimidines

Thymidine and cytidine

Purines

Adenosine and guanosine

Chargraff Experiments

• Showed that amounts of A = T and C = G

• Percentage of C + G does not necessarily equal the amount of A + T

Watson and Crick (1953)

• Proposed that DNA is a right-handed double helix

• Strands run antiparallel to each other and their bases are stacked

• A-T and C-G base pairing connect the strands

• 10 base pairs per helix turn

Strand complementarity

• Caused by A-T and C-G pairing

• Adds chemical stability to the double helix

  • A-T forms 2 H bonds

  • C-G forms 3 H bonds

Central Dogma of Molecular Biology

DNA → RNA → Protein

Semi-Conservative Replication

• How DNA actually replicates

• The complementarity of DNA allows for one strand to serve as a template

• Each replicated DNA strand has one old strand and one new strand

Conservative Replication

The original helix is preserved, and two newly synthesized strands come together

Dispersive Replication

Parental strands are distributed into two new double helixes

Meselson and Stahl Experiments (1958)

• Used labeled bacteria to determine how DNA replicates

• Found that DNA replication is semi-conservative in prokaryotes

• Centrifuged bands showed persistence of originally marked DNA

Bacterial DNA Replication

• Circular, originating from a single point

• Bidirectional - Two replication forks

Replication Fork

• Structure in DNA replication

• Created where the strands of DNA are unwound

DNA Polymerases

• Catalyze DNA synthesis

• Requires:

  • DNA template

  • Four deoxyribonucleoside triphosphates (dNTPs)

  • DNA or RNA primer

Nucleosides

• DNA bases with a P-P-P structure that provides energy for bonding

• How nucleotides arrive at the replication fork

• Will be bonded to the growing strand by DNA polymerase

DNA Chain Elongation

• Occurs in the 5’ to 3’ direction

• Proceeds by the addition of one nucleotide at a time to the 3’ end

• As nucleotide is added, terminal phosphates are cleaved to make space for a new nucleotide

Bacterial DNA Polymerase III

• Responsible for 5’ to 3’ elongation in prokaryotes

• Essential to replication

Helicases

• Unwind the DNA helix

• DnaA binds to the origin of replication and begins unwinding

• DnaB and DnaC further open and destabilize the helix

• Single-stranded binding proteins (SSBPs) stabilize the open conformation

Toposoimerases

• Prevent DNA in front of the replication fork from becoming too tightly wound as the DNA opens up

DNA Sliding Clamp

• Helps DNA polymerase move along the template without falling off

• Loaded onto DNA by clamp loaders

RNA Primer

• Aids DNA Polymerase III in chain elongation

• Primer is removed by DNA Pol III and replaced with DNA

Leading Strand of DNA

• Synthesized continuously in the 5’ to 3’ direction

Lagging Strand of DNA

• Synthesized in discontinuous Okazaki fragments

• Each fragment has its own RNA primer

  • DNA polymerase I removes these primers

  • Fragments are joined by DNA ligase

DNA Proofreading

Two strands are run through the DNA polymerase molecule after replication to catch any errors

Eukaryotic DNA Replication

• More complex than prokaryotic because

  • There is more DNA

  • Chromosomes are linear

  • DNA is complexed with proteins

  • Multiple origins of replication on chromosomes

Telomeres

• The ends of chromosomes

• Provide structural integrity

• Consist of long stretches of repeating sequences

• Problematic to replicate, telomerase required to counteract the shortening of telomeres

Telomerase

• Consists of

  • Telomerase reverse transcriptase (TERT)

  • Telomerase RNA (TR)

• TR provides a template for a repeating sequence

• TERT inserts the new DNA sequence

Chromatin

• Organization structures of Eukaryotic chromosomes

• Condenses to become visible chromosomes during DNA replication

• Types

  • Euchromatin

  • Heterochromatin

Euchromatin

• Uncoiled and active chromosomes or regions

• Most areas of chromosomes in active cells

• Usually areas where gene expression is occurring

Heterochromatin

• Condensed and inactive chromosomes or regions

• Inactive because they either lack genes or contain genes that are repressed

• Ex: Telomeres and centromeres

Histone Proteins

• Abundant molecules with highly conserved sequences in eukaryotes

• Provide the first level of packaging for a chromosome

Nucleosomes

• 146 Base pairs of supercoiled DNA

• Wound around a core of eight histone molecules

Levels of DNA Packaging

Organization is around a central scaffold

1. 2 nm double-stranded DNA molecule

2. 11 nm nucleosomes

3. 30 nm chromatin fiber

Histone Core

• Two copies of H2A, H2B, H3, and H4 in an octamer

• H1 resides outside of the core

  • Linker histone that binds to linker DNA

  • Connects one nucleosome core particle to the next

2nm DNA Organization Structure

Double Stranded DNA Molecules

11 nm DNA Organization Structure

• Nucleosomes

• “Beads on a string” form of chromatin

• Produced in the first level of packing

30 nm DNA Organization Structure

• Method of compaction unknown, but H1 is vital

• Produced in the second level of packing

300 nm DNA Organization Structure

• Compaction continues by looping the 30 nm structures and attaching them to nonhistone protein scaffolds

• Looped DNA attached to the nuclear matrix via MARS

Nonhistone Proteins

• Other proteins that are associated with the chromosomes

• Amount and types vary per cell

• May have a role in compaction or something else related to the DNA

The SWI/SNF Complex

• ATP-dependent chromatin remodeling complex

• Switching (SWI) and sucrose non-fermenting (SNF)

• 9-12 subunits

• Each subunit required for function of entire complex

• Evolutionarily conserved

Histone Modifications

• Covalently attached groups to histone tails

• Reversible

• Dynamic

• Have diverse biological functions

Histone Tails

• Have regulatory roles

• Acetylation

  • any lysine (gene activation) K

• Methylation

  • lys9 (gene repression K9

  • lys4 (gene activation) K4

  • lys36 (transcription elongation) K36

Histone Acetylation

Activates transcription, reducing ability of nucleosomes to repress it

Chromatin Immunoprecipitation (ChIp)

Detect histone modifications

Histone Methylation

• Can occur on Arg (R) or Lys (K) residues, with selectivity for lys

• Condenses chromatin into heterochromatin

Epigenetics

• The study of heritable changes of DNA that don’t involve changes in DNA sequence

• Histone tails

• DNA Methylation

• CpG Islands

• Imprinting

DNA Methylation

• Maintains a gene in an inactive state

• Involved in the regulation of many processes

CpG Islands

• Long stretches of DNA that are CpG rich

• Located in promoter regions, unmethylated to allow transcription

Genomic Imprinting

• A form of epigenetic inheritance

• Regulation of the gene depends on the sex of the transmitting parent

Transcription

• The process of creating RNA by copying part of the DNA sequence into a complementary sequence

• RNA Polymerase binds, separates DNA strands, and uses on eDNA strand as a template

• Initiation → Elongation → Termination

RNA Synthesis Differences from DNA Synthesis

• No primer needed

• ribonucleotides instead of deoxyribonucleotides

  • ribonucleoside triphosphate precursors

• Only one strand of DNA required

• Uridine replaces Thymidine

Messenger RNA (mRNA)

Transfers DNA code to ribosomes for translation

Transfer RNA (tRNA)

Brings amino acids to ribosomes for protein synthesis

Ribosomal RNA (rRNA)

Ribosomes are made of rRNA and protein

mRNA Components

• 5’ Untranslated Region (UTR)

• The coding sequence (open reading frame)

• 3’ Untranslated Region (UTR)

Open Reading Frame

• Coding sequence of RNA/DNA

• Specifies the amino acid sequence of the protein that will be synthesized during translation.

• Varies in length according to the size of the protein it encodes

RNA Polymerase Alpha Subunit

Assembles the tetramatic core

RNA Polymerase Beta Subunit

Ribonucleoside triphosphate binding site

RNA Polymerase Beta-Prime Subunit

DNA template binding region

RNA Polymerase Sigma Subunit

Initiation of transcription

Transcription Start Site (TSS)

• Where transcription begins

• DNA double helix is unwound to make the template strand accessible to RNA polymerase

• Occurs downstream from promotors (like the TATAAT box)

Rho-dependent Termination

• Transcription termination method

• Protein factor “Rho” binds the RNA and destabilizes the interaction between the template strand and mRNA

• New mRNA is released from elongation complex

Rho-Independent Termination

• Transcription termination method

• RNA transcription stops when the newly synthesized RNA molecule forms a hairpin loop followed by a run of Us

• RNA destabilizes and detaches from the DNA

Chromatin Remodeling

• Required for eukaryotic transcription

• Uncoils chromatin, making DNA accessible to RNA polymerase

• AKA Acetylation (H3K4, H3K9)

Promoters

• Regions of DNA that ‘promote’ the transcription of the genes they regulate

• Located upstream of regulated genes, on the same strand

RNA Polymerase I

Produces: rRNA

Location: Nucleolus

RNA Polymerase II

Produces: mRNA, snRNA

Location: Nucleoplasm

RNA Polymerase III

Produces: 5s rRNA, tRNA

Location: Nucleoplasm

Transcription Factors

• Aid RNA polymerase in interacting with promoters

  • Necessary because RNA polymerase can’t bind directly to promoters

• Recognize and initiate transcription at specific promoter sequences

• DNA-Binding Domain and Activation Domain

General Transcription Factors

Required for all RNP II-mediated transcription

TATA Box

• Core promoter element

• Binds the TATA-Binding Protein (TBP)

• Determines the start site of transcription

Enhancers and Silencers

• Can be upstream, within, or downstream of a gene

• Can modulate transcription from a distance

• Increase or decrease transcription in response to a cell’s need for a gene product

5’ Cap

• Modified guanine structure added to the 5’ end of all mRNAs

• Only on RNA transcribed from RNA Pol II

• Aids in

  • Transport

  • Protection

  • Activity

Poly(A) Tail

• Added to the 3’ end of mRNA

• Cleavage and Polyadenylation Specific Factor cleaves the RNA so this structure can be added

RNA Splicing

• Spliceosomes remove introns and join together exons

• Needed so mRNA can produce the correct proteins during translation

Spliceosome

• Removes introns during splicing

• Consists of non coding RNA (U snRNAs) and U snRNA-specific proteins (U snRNPs)

The genetic code is

• Composed of nucleotide triplets that specify amino acids (codons)

• Non-overlapping. Triplets are read in order

• Unambiguous. Each codon specifies a certain amino acid and only one.

Genetic code degeneracy

• Some amino acids are specified by more than one codon

• One start codon (ATG or AUG) and 3 stop codons

• 20 amino acids but 64 codon combinations

Translation

The polymerization of amino acids into polypeptide chains.

Requires:

• mRNA

• ribosomes

• tRNA

• amino acids

tRNAs in Translation

• Have anticodons that complement the mRNA codons

• Each tRNA carries an amino acid corresponding to that anticodon

• Charged (linked to amino acids) aminoacyl tRNA synthetase

Amino Acids

• Consist of:

  • Carboxyl group

  • Amino group

  • R (radical) group

• Joined together by peptide bonds

Prokaryote Ribosomes (70S)

Large subunit: 50S

Small subunit: 30S

Eukaryote Ribosomes (80S)

Large subunit: 60S

Small subunit: 40S

Ribosome Binding Sites

• A Site (Arrival)

• P Site (Peptide)

• E Site (Exit)