Chpt 4 Flashcards
Central Dogma of Molecular Biology
-What part is replication, Transcription, and Translation?
DNA->RNA->Protein
Replication= DNA to DNA or DNA directed DNA synthesis
Transcription=DNA to RNA or DNA directed RNA synthesis
—>Processing of mRNA capping, polyadenylation, splicing
Translation=RNA to Protein or RNA directed protein synthesis
Where does replication, transcriptions and translation take place? (In a eukaryotic cell)
Eukaryotic cell has a nucleus
Nucleus:
-replication
-transcription
Cytoplasm:
-Translation
Functions of Nucleic Acids (3)
Building blocks of DNA and RNA
- DNA=Genetic Material
- RNA=Adaptor molecule between DNA and protein
Transport chemical energy within the cell
-ATP
Signal Molecules
-Cyclic AMP
Nucleic Acids
-linear, non branched polymer of nucleotides
What are the classes of nucleic acids?
1) RNA=ribonucleic acid
2) DNA= 2’ deoxyribonucleic acid
What does. a nucleotide contain?
- Pentose sugar
- Nitrogenouse base
- Phosphate (one or more)
What are the nucleotides? and the two classes of nucleotides?
Pyrimidines:
Thymine (T)
Cytosine (C)
Uracil (U)
Purines:
Adenine (A)
Guanine (G)
Thymine
Pyrimidine Base
5-methyl-2,4-dioxypyrimidine
DNA only
Cytosine
Pyrimidine Base
4-amino-2-oxypyrimidine
DNA and RNA
Uracil
Pyrimidine Base
2,4-dioxypyrimidine
RNA only
Adenine
Purine
6-aminopurine
DNA and RNA
Guanine
Purine
2-amino-6-oxypurine
DNA and RNA
Sugar Phosphate Backbone
Nucleotides connected by 3’ to 5’ phosphodiester bond
Imparts uniform negative charge to DNA/RNA
- negative charge repels nucleophilic species (ex hydroxyl) thus the phosphodiester bond resists hydrolytic attack
- Seperation by agarose gel electrophoresis
Creates 3’ and 5’ end (directionality)
-nucleotide sequences are written 5’ to 3’: L to R
How are bases attached to sugars?
Beta Glycosidic linkage
Nucleotide vs Nucleoside
Nucleoside:
sugar + nitrogenous base
Nucleotide
sugar + nitrogenous base + phosphate
What data did Watson and Crick use to determine the structure of DNA
- xray diffraction photograph of DNA crystals
- Chargraff’s rule
- Bond Angles from reference books
- Built models
Chargraff’s rule
Edwin Chargraff determined the composition of DNA from many organisms
- [A]=[T]
- [G]=[C]
rules:
- the four nucleotides are not present in equal amounts
- relative ratios of the four bases are not random, and vary from one species to another
- A=T and G=C
X-ray diffraction photograph of DNA crystals
Maurice Wilkins and Rosalind Franklins
-two chains that wind in a regular helical structure
DNA is a HELIX- 3.4 A spacing
MP of DNA factors on?
nucleotide content determines melting point of DNA (# of h bonds)
G to C = 3 bonds
A to T= 2 bond
Who received the Nobel Prize in Physiology or Medicine in 1962?
Francis Crick
James Watson
Maurice Wilkins
DNA!
What holds DNA together?
Hydrogen bonding between base pairs
Hydrophobic interactions (Van Der Waals) due to base stacking
B form of Double Helix
Normal Form-Watson and Crick Form
Diameter of Helix- 20 A
10.4 Base pairs per turn
1 Base pair is 3.4 A
Characteristics: Complementary base pairing Major Groove Minor Groove Antiparallel Hydrogen bonding between complementary BP
A form of Double Helix
- dehydrated B form
- nucleotide tilted 20 degrees relative to helical axis
Z form of Double Helix
Zig Zag Form
stretches of alternating purine and pyrimidine
-base pairs flip 180 degrees
-left handed helix
what is DNA organized into?
Gene
Gene
- discrete functional unit of DNA
- when expressed (transcribed), yields a functional product->rRNA, tRNA, snRNA, and mRNA is translated into a polypeptide sequence (protein)
- open reading frame->long stretch of nucleotides that can encode polypeptide due to absence of stop codons
Karyotype
- Photograph of chromosomes from a single organism
- Arranged by size (Largest #1 to smallest #22)
- Homo sapiens- 46 chromosomes, 23 pairs
What are the parts of a chromosome
- Centromere
- Kinetochore
- Telomere
Centromere
Site that connects sister chromatids
Kinetochore
attachment site of spindle to chromosome
Telomere
nucleotide repeat at the end of linear chromosome
- TTAGGG x 1000
- synthesized by telomerase
Properties of DNA
- Melt/Anneal/ Reanneal
- Hypochromic effects
- Supercoiled/Relaxed
Double Stranded DNA and Heat
Double stranded DNA can reversibly melt
- Heating DNA breaks hydrogen bonds between base pairs (acid or base also works)
- At melting Temperature half of the helical structure is lost
- Single stranded DNA absorbs light more efficiently than double stranded
Hypochromic Effect
or Hypochromism
- DNA can melt than reanneal
- If sequences are similar they will reanneal or hybridize
In what organisms are DNA circular and what organisms are there DNA linear?
Prokaryotic, mitochondrial, and chloroplast genomes are circular
-circular molecules may exist in topological isomers (relaxed and supercoiled)
Eukaryotic genomes are linear
Single Stranded Nucleic Acids (DNA or RNA) can form complex structures
Stem loops
- are produced by H-bonding between complementary regions in DNA and RNA
- H-bonding stabilizes more complex structures
- mismatches are observed
- often observed in ribosomal RNA molecules
Testing the Semiconservative Replication Hypothesis
-Grew E. coli in 15NH4Cl until DNA was completely labeled
-transferred E. coli to 14NH4Cl containing media
-Followed labeling pattern of DNA through several generations using density gradient equilibrium sedimentation
By 4 Generations you get Density 14, Hybrid and Density 15 gene
DNA replication: DNA polymerase
- adds deoxyribonucleotide units to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction
- Requires: Four dNTPs- (dATP, dGTP, dCTP, dTTP)
- Divalent Cation (Mg2+)
- Template DNA
- Primer provides 3’ OH
DNA polymerase Rxn mechanism
Nucleophilic attack by the 3’ OH on the alpha phosphate group of dNTP
-PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)
Types of RNA
- ribosomal RNA (rRNA)- part of the ribosome
- transfer RNA (tRNA)
- messenger RNA (mRNA)- sequence translated into protein sequence
- small nuclear RNA (snRNA)- involved in splicing (spliceosome)
- micro RNA (miRNA)- small RNA complementary of mRNA that inhibits translation of mRNA
- small interfering RNA (siRNA)- small RNA that binds to mRNA causing destruction of mRNA
Transcription: RNA polymerase
-adds ribonucleoside triphosphate units to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction
requires:
- four NTPs (A, U, G, C)
- Divalent Cation (Mg2+)
- Template DNA
- no primer needed
- lacks endo and eco nuclease activity
What are the RNA polymerase’s for Eukaryotic and prokaryotic cells
Prokaryotic polymerase (1)- RNA polymerase
Eukaryotic polymerase (4)
- RNA poly I
- RNA poly II
- mRNA
- RNA poly III
RNA transcribing
Genes may or may not be transcribed depending on the needs of particular cell type
- gene is a functional region of DNA
- expressed genes are TURNED ON
- unexpressed genes are TURNED OFF
RNA polymerase Rxn Mechanism
Nucleophilic attack by the 3’ OH on the alpha phosphate group of NTP (ribonucleoside triphosphates)
-PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)
mRNA relationship to Template Strand and coding strand of DNA
mRNA is complementary to template strand
mRNA is identical (except for U to T changes) to the coding strand
Prokaryotic Promotor Site
Pribnow box (also called TATA box)
- 5’ TATAAT 3’ centered -9/-10
- designated by the 5’ to 3’ sequence on the NONtemplate strand–> 8 to 10 nucleotides left (5’ or upstream) of transcriptonal start site (designated +1-there is no 0 nucleotide)
-35 sequence
5’ TTGACA 3’
Eukaryotic Promotor
Class II genes
-those synthesized by RNA poly II (pre mRNA and snRNA)
Parts
- TATA or Hogness box
- GC box (GGGCGG)
- CAAT box
Transcriptional Termination
Rho dependent
-involves protein called RHO
Rho independent
- involves stem loop structure in mRNA
- stem loop is followed by UUUs
prokaryotic and eukaryotic mRNA
prokaryotic mRNA are polycistronic
-may encode two or more proteins
eukaryotic mRNA are monocistronic
-encode only one protein
eukaryotic mRNA post transcriptionally modified
Capping
-attachment of 7-methylguansine using 5’ to 5’ triphosphate linkage
Polyadenylation
-attachment of 40 to several hundred adenine nucleotides to 3’ end of mRNA
Splicing
-removal of introns
Amino Acids and tRNA
Amino acids are attached to 3’ end of tRNA
-Aminoacyl-tRNA synthetase–> attach amino acid to tRNA
Stages of Translation
Initiation
-assemble and align ribosome, mRNA, and tRNA^fmet
Elongation
-template directed synthesis of proteins
Termination
- termination factors halt protein synthesis
- ribosome, mRNA, and new protein dissociate
Orientation of Translation
- Ribosomes move 5’ to 3’ along mRNA
- protein is synthesized N to C
Genetic Code
Specific Unambiguous
- specific codon always codes for SAME amino acid
- three nucleotides (codon)=one amino acid
Universal
- conserved from species to species
- main exception=mitochondria
Redundant (also called degenerate)
-amino acid may have more than one codon
Nonoverlapping and comma less (no punctuation)
- read from fixed starting point (AUG)
- lacks punctuation between codons
Translation Start Site
AUG encodes Met (n-terminal amino acid)
- Prokaryotes use a Shine-Dalgarno sequence to align a ribosome on the mRNA upstream (5’) of AUG
- eukaryotes use the 5’ cap to align the ribosome on the mRNA
Eukaryotic mRNA contain Exons and Introns
Exons-coding region
Introns- (intervening regions) nocoding regions
How were Introns discovered?
introns were discovered by hybridizing mRNA to genomic DNA
Splicing
removal of introns
- spliceosome- specific proteins and small nuclear RNA
- most introns start with GU and end with AG
