Genetics Exam 3

Friedrich Miescher

Friedrich Miescher

First isolated DNA from white blood cells

Albrecht Kossel

Determined that DNA has 4 nitrogenous bases

Phoebus Levene

Discovered DNA is made of nucleotides (phosphate group, sugar and a base) and proposed that tetra-nucleotide structure of DNA

Griffth

Demonstrated the phenomenon of transformation, but did not identify transforming principle

Avery, Macleod, and McCarthy

Identified DNA as a transforming principle

Hershey and Chase

Identified DNA as the genetic material in bacteriophages

Heinz Fraenkel-Conrat and Bea Singer

Discovered that RNA is the genetic material of Tobacco Mosaic Virus

Yanofsky

Established that changes in DNA and protein were collinear (similar in location of changes)

Rosalind Franklin and Maurice Wilkins

Used x-ray diffraction to conclude that DNA was helical

Erwin Chargaff

Determined that DNA for yeast consisted of an equal amount of purines and pyrimidines - therefore concluding the base compositions and that A=T and G=C

James Watson and Francis Crick

Published the structure of DNA

Francis Crick

Proposed non-overlapping code

Brenner and Crick

Established codon consisted of 3 non-overlapping nucleotides

Meselson and Stahl

Grew E. Coli in heavy nitrogen for many generations to the transfer the cells to light nitrogen to replicate and determined DNA replicates through semiconservative replication

Fire and Mello

Learned that genes with nucleotide sequence that was same as injected dsRNA in worms had decreases expression, allowing them to determine that RNAi limits invasion of foreign genes and censors the expression of cell’s own growth

Charpentier and Doudna

Were first to use CRISPR as a “cut and paste” tool to edit gene sequences

Nirenberg and Matthaei

Were first to determine that a specific RNA sequence coded for a specific amino acid by using Poly-U, cell-free extracts, and 20 amino acids where 1 is radioactive. By determining if the protein is radioactive, they could conclude that the labeled amino acid was in the protein and the codon codes for that

Nirenberg and Leder

Used tri-nucleotide, ribosome, and 20 charged tRNA’s (1 radioactive amino acid) to identify 61 different codons

Nucleoside

Sugar and Base

Nucleotide

Sugar, Base, Phosphate

Euchromatin

Lighter staining parts of the chromosome during interphase

• Actively transcribed gene

• Condenses and relaxes

Heterochromatin

Darker staining parts of chromosome

• Fewer Genes

• Usually condensed

• Usually not involved in crossing over

• Replicated in S phase

Constitutive vs Facultative Heterochromatin

• Constitutive: always heterochromatin

• Facultative: May be Euchromatic sometimes (X chromosome that is Barr Body)

Histones

Basic proteins that are composed of positively charged amino acids (highly conserved)

Nucleosome

Nucleosome core + ~53 base pairs linker DNA

Nucleosome Core

Core histones + ~147 base pairs DNA

Core Histones

Composed of 2 each of H2A, H2B, H3, H4

Positive Supercoiling

Over-rotated in same direction as DNA so left-handed supercoil compensates

Negative Supercoiling

Under-rotated in same direction as DNA so right-handed supercoil compensates

Topoisomerase

Alters torsional stress in DNA by cutting DNA backbone to rewind

Endopolyploidy

Several rounds of DNA replication without separation of replicated chromosomes

Puffs

Areas where the DNA is loosely coiled so that transcription can occur

Theta Replication

Common in bacteria and other circular DNA molecules where Replication proceeds in both directions from origin (bidirectional).

Rolling Circle Replication

the F factor and some viruses (lambda) where Replication continues around many times producing many strands that are used as templates to synthesize many double stranded circular DNA molecules

Linear Eukaryotic Replication

For Eukaryotic Chromosomes which have multiple origins for replication (bidirectional)

In which direction are nucleotides added by DNA Polymerase?

5’ to 3’

DNA Polymerase I

5’-3’ exonuclease activity to remove and replace RNA primers, proofreads with 3’ to 5’ exonuclease activity

DNA Polymerase II

DNA repair and restates replication after damage halts synthesis

DNA Polymerase III

Elongates DNA and restates replication after damage halts synthesis, proofreads with 3’ to 5’ exonuclease activity, adds nucleotides to the primer

DNA Polymerase IV

DNA repair

Okazaki Fragments

Fragments of newly synthesized DNA created on the lagging strand

Initiator Protein

Binds to the origin of replication causing local unwinding and short stretch of single-stranded DNA

Helicase

Attaches at replication fork and moves into the fork breaking H-bonds

Single-Stranded Binding Proteins

Coat single-stranded DNA to protect it, stabilize it, and prevent double-strand DNA (hairpins)

Gyrase

A topoisomerase that releases supercoiling ahead of replication fork as it unwinds

RNA Primers

Made by Primase (an RNA polymerase)

Primosome

Helicase and Primase

DNA Ligase

Seals the nicks (missing sugar-phosphate bond)

Replication Licensing Factor

Attaches to each origin of replication early in cell cycle

Telomerase

Ribonucleoprotein that contains RNA which it uses to make several repeats of DNA to extend the 3’ end of the telomere

RNA Polymerase I

large rRNA

RNA Polymerase II

mRNA, snRNA, snoRNA, miRNA

RNA Polymerase III

small rRNA, tRNA, snRNA

Ratl Exonuclease

Chews up RNA (exonuclease) and removes nucleotides from DNA

• Cap on 5’ to prevent it from chewing up mRNA that is needed

RNAi = RNA interference

Mechanism not RNA type

Shuts off gene expression using dsRNAs in eukaryotes

hnRNA

Heterogenous RNA, Pre-mRNA, Precursor for mRNA, Primary transcript for mRNA

• Addition of 5’ cap

• 3’ Cleavage and addition of Poly-A-tail

• RNA splicing

• RNA editing

• Internal RNA modifications

rRNA = Ribosomal RNA

Most abundant

Structural and functional components of the ribosome

Ribosomes have two subunits and it is not fully assembled until transcription

Precursor rRNA

Contains all rRNAs except 5S rRNA in eukaryotes

snoRNA = Small Nucleolar RNA

Processing and assembly of rRNA

tRNA = Transfer RNA

• Help incorporate amino acids into polypeptide chain

• Amino acid site is always CCA

• Ribthymine: thymine with ribose attached

• Pseudouridine: ribose attaches to 5 position of ring instead of 1 position

• Inosine: derivative of guanine

• A site: Aminoacyl

  • In P and A sites peptide bond is forming between amino acid

• P site: Peptidyl

  • tRNA releases amino acid

• E site: Exit

snRNA = Small Nuclear RNA

Processing pre-mRNA

Regions complementary to exons

snRNPS = Small Nuclear Ribonucleoproteins

Critical in proper positioning for splicing and base-pairing is important in their binding to RNA

gRNA = Guide RNA

Aid in insertion and deletion types of editing

dsRNA = Double Stranded RNA

Can form miRNA and siRNA using enzyme dicer to leave dsRNA

miRNA = MicroRNA

Inhibits translation of mRNA

Transcribed from distinct gene and targets other genes for regulation

siRNA = Small Interfering RNA

Triggers degradation of other RNA molecules

Comes from mRNA, transposons, or viral RNA and targets the genes that it comes from

RISC

miRNA and siRNA bind with proteins to form an RNA induced splicing complex that base pairs with mRNA and either inhibits translation (miRNA) or degrades mRNA (siRNA)

piRNA = Piwi-Interacting RNA

Suppresses the transcription of transposable elements in reproductive cells

Protects germline cells from transposons by silencing transposon RNA

lncRNA = Long Non-Coding RNA

Variety of functions

Decoys to bind regulatory proteins or miRNAs, pair with mRNA to trigger RNAi, Xist RNA recruit proteins to inactive X-Chromosome

crRNA = CRISPR RNA

Only in prokaryotes

Assists destruction of foreign DNA

Cas9 protein is a special enzyme that binds with it to cut the DNA

Aminoacyl Synthetase

Seen in each amino acid and can recognize all tRNAs for that amino acid - helps pair amino acid to its tRNA

Shine-Dalgarno Sequence

Located in the 5’ untranslated region to help with ribosome binding

*translation

Polycristronic mRNA

1 mRNA molecule produced, but contains the information for more than one gene and is translated into more than 1 gene product

• mRNA always reads 5’ to 3’ end

• Ribosome binds at 5’ end

• Eukaryotes are not polycistronic

Peptidyl Transferase Activity

Ribozyme forming peptide bond between amino acids

Polysomes

Multiple ribosomes translating the same mRNA simultaneously

Splicosome

SnRNPs and pre-mRNA complex structure at which introns are removed and exons are joined together

Methylation

Occurs and identifies separate rRNA-to-be molecules

What are some differences in Eukaryotic Replication vs Prokaryotic?

• Have many origins

• Origin must be approved for replication

• Linear chromosomes

• Many DNA polymerase molecules (only DNA polymerase I and III in prokaryotes)

• Have telomeres which replicate

• Nucleosome assembly immediately follows replication

Transcription Unit

A segment of DNA that codes for an RNA molecule and the sequences necessary for its transcription

Consensus Sequence

A sequence that describes the nucleotides most often present in a segment of interest

• Y = Pyrimidines

• R = Purines

• N= Any

Upstream

Sequences prior to the start of transcription

Downstream

Sequences after the start of transcription (+1 and up)

Core enzyme

a2BB’ (core RNA polymerase) (B = beta)

Holoenzyme

a2BB’ o (B = beta and o = sigma)

Rho (p)

Binds to the RNA and moves toward 3’ end and RNA polymerase, its helicase activity causes the DNA-RNA hybrid to unwind and transcription ends

Cis Elements

Same piece of DNA, adjacent to the genes they will regulate; cannot move

Trans Elements

Can move around and interact with many genes, attach to cis-elements or other transcription factors, and recruit RNA polymerase

Core (basal) promoter

Immediately upstream of gene and where the basal transcription apparatus binds (contains TATA box)

Regulatory Promoter

Immediately upstream of core promoter; variety of consensus sequences

3 Types of Processing

• Capping of 5’ end

• Poly-A-tail added to 3’ end

• Removal of introns

Primary Structure

Amino acid sequence

Secondary Structure

Chain of amino acids folding on itself (alpha helix and beta pleated sheet)

Tertiary Structure

Protein folding secondary structure

Quaternary Structure

Protein containing more than one subunit - the joining of subunits

Wobble Effect

Single tRNA can pair with more than one codon

• Inosine can pair with U, C, and A

• Guanine and Uracil can also pair