Genetics Exam 3

First Purification of a Nucleic Acid

By Freidrich Meiser

Seperated nuclei from the cells of WBCs and called it “nuclein”

Nuclein

Contained large amounts of phosphorus, but no sulfur

Meaning it was not a protein

Now known as DNA

Levene’s Tetranucleotide Hypothesis

Incorrect

Suggested that DNA was made of short repetitions of the same four nucleotides

Assumed A, C, G, and T were all present in equal proportions

Early Ideas about the Unknown Genetic Material

1. Capable of replication

2. Capable of storing information

3. Complex enough to generate thousands of gene products

4. Any changes in it (mutations) should lead to phenotypic changes

The Griffith Experiments

Demonstrated bacterial genetic transformation (Streptococcus pneumoniae)

Type IIIS: virulent

Type IIR: non-virulent

Mice injected with heat-killed IIIS and live IIR died

Due to an unknown “transforming principle”

Transformation

A herritable change in a cell or organism brought about by exogenous DNA

Avery, MacLeod, and McCarty Experiments

Identified the “transforming principle” to be DNA

Heat-killed IIIS were treated with either RNase, Protease, or DNase

Only cultures treated with DNase could no longer transform IIR bacteria into IIIS bacteria

Hershey and Chase Experiments

Showed that DNA is the infectious agent in bacteriophages

1. Infect bacteria in radioactive media with T2 phages

32P phages have radioactive DNA, 35S phages have radioactive proteins

2. Infect cells in non-radioactive media with those radioactive phages

3. Separate the phages from the cells

4. After infection, separate the empty phages (“ghosts”) from the cells

X-Ray Diffraction

1. Crystals of a substance are bombarded with X-rays which are diffracted

2. The spacing of the atoms in the crystal determines the diffraction pattern, which shows up as spots on a photographic film

3. The doffraction pattern provides info about the structure of the molecule

Rosalind Franklin

Did x-ray crystallography of DNA fibers in Maurice Wilkins’ lab

Diffraction pattern showed a helical structure, possibly a double helix, with a sugar-phosphate backbone

Chargaff’s Rules

The proportion of A equals the proportion of T, and the proportion of C equals the proportion of G

There is an equal proportion of purines (A and G) to pyrimidines (T and C)

The proportion of C+G does not necessarily equal that of A+T

The Watson-Crick Model

DNA is a double helix with antiparallel strands

Pentose sugar and phosphate backbone

Nitrogenous bases paired at the center by H bonds

Alternating major and minor grooves

One while turn of the helix is about 10 bp

Ribose vs, Deoxyribose

Deoxyribose lacks the 2’ OH group

Purines

Adenine and Guanine

Double carbon and nitrogen aromatic ring stuctures

Pyrimidines

Thymine, Cyrosine, and Uracil

Single carbon and nitrogen aromatic ring structures

Nucleoside

sugar + a nitrogenous base

Nucleotide

sugar + a nitrogenous base + phosphate(s)

Ribonuclosides

Adenosine

Cytidine

Guanosine

Uridine

Add: monophosphate, diphosphate, or triphosphate

Deoxyribonucleosides

Deoxyadenosine

Deoxycytidine

Deoxyguanosine

Deoxythymidine

Add: monophosphate, diphosphate, or triphosphate

Phosphodiester Bond

Between two nucleotides

Phosphate group joins the 3’ carbon of one nucleotide and the 5’ carbon of the other through two ester linkages:

A C-3’ to C-5’ bond

Result: a dinucleotide with 5’ to 3’ polarity

Adenine-Thymine

Two hydrogen bonds form between them

Also between adenine and uracil in RNA

Guanine-Cytosine

Three hydrogen bonds form between them

Higher density and higher melting point

Intramolecular Hydrogen Bonds in Transfer RNA

Most RNAs are single stranded, so intramolecular hydrogen bonds can form

Result: endless possibilities for 3D structures

Organization of DNA in Eukaryotic Nucleus

Chromosomes are always present, but only visible durong cell division

Nucleosome is the basic unit of organization

Nucleosomes

Two turns of DNA wrapped around a cluster of 8 histone proteins and held by a 9th (histone 1)

Adjacent nucleosomes pack together to form 30 nm chromatin fibers

Exonucleases

Enzymes that remove nucleotides one by one from the ends of a polynucleotide chain

Endonucleases

Enzymes that cleave phosphodiester bonds within a polynucleotide chain

Restriction Endonucleases

Recognize a specific base pair sequence, called a restriction site, with a palindrome sequence and makes a cut there

Restriction Fragments

Due to restriction digestion

Smaller linear DNA fragments with either complementary single-strand cohesive tails (sticky ends) or blunt ends

Depends on which restriction enzyme

Electrophoresis

Seperates DNA fragments based on size

Smaller fragments move farther faster

Negative charges in the phosphate groups cause DNA fragments to move to positive end

Watson-Crick Model for the Semiconservative Replication of DNA

Parental strands separate and serve as templates for new, complementary strands

Each new DNA molecule contains one parent strand

Density Gradient Centrifugation

Separates molcules by density using high speed centrifugation of a heavy salt solution

Using CsCl generates a CsCl density gradient: lower density at the top of the tube, higher density at the bottom

DNA with the N15 isotope have a higher density than DNA with N14 (the most abundant form)

Meselson and Stahl Experiment

The most beautiful experiment in biology; proved semiconservative replication

Grew E. coli in a N15 medium so they have high density DNA (Gen 0)

Transfer them to a N14 medium and allow them to divide once (Gen1) will have intermediate density

Allow cells to divide a second time (Gen 2) will have 50% intermediate DNA and 50% light DNA

Replication of Circular Chromosomes in Bacteria

oriC is the only origin of replication site

It is recognized by an initiator protein (Dna A in E. coli) that opens 1 replication bubble with 2 replication forks

ter: replication termination site

self-replicating DNA molecules

Replication of Linear DNA in Eukaryotes

Multiple origins of replication, where replication bubbles form

Each bubble has 2 replication forks

Eventually, forks of adjacent bubbles run into each other, and the segments of DNA fuse

DNA synthesis must be antiparallel

Catalyzed by DNA polymerase:

DNA polymerase

Adds nucleotides to the 3’ end of the growing new strand

Known as 5’ to 3’ polymerase activity

Two needed per replication fork: one for each strand

Formation of the Phosphodiester Bond

New DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs)

Incoming nucleotides must be in the triphosphate form

Hydrolysis of a phosphoanhydride bond between phosphates releases the energy needed to form the new phosphodiester bond

Proofreading Activity of DNA Polymerase

3’ to 5’ exonuclease activity

If polymerase places a wrong nucleotide, it can go back, remove it, and continue on

Still makes mistakes sometimes: 1 mistake per 10^7 nucleotides copied

Leading and Lagging Strands

Defined for each fork, not for the entire bubble

Lagging strand: discontinuous replication/Okazaki fragments

DNA Helicase

Unwinds the strands after they are separated

Single-Stranded DNA Binding Proteins (SSBPs)

Bind to the separated DNA strands to stabilize the open conformation

Gyrase

Unwinds supercoiled DNA ahead of the replication fork

In bateria: makes single strand cuts, allows it to unwind, reseals the cut

Sliding Clamp

Prevents polymerase from falling off

DNA Polymerase III

Main polymerizing agent in bacteria

Extends DNA using RNA primers

Can only extend an existing strand of nucleic acid, can’t initiate a new strand from scratch

Primase

Makes a small RNA primer that DNA polymerase adds deoxyribonucleotides to

Removed by DNA polymerase I

DNA Polymerase I

Only DNA polymerase with 5’ to 3’ exonuclease activity

Removes primers then replaces it with DNA

Then ligase seals it

Chromosome Shortening in Linear DNA Replication

The ends of the DNA strands have gaps left due to the removal of primers

The gaps can’t be filled because there is no 3’ template for a primer

Prevented with telomerase

Telomerase

A ribonucleoprotein that serves as a guide and template for the synthesis of complementary DNA (cDNA)

Present in: single cell eukaryotes, stem cells in animals/plants, germ cells, cancer cells, and activated lymphocytes

Telomerase Activity in Tetrahymena

1. Telomerase guide RNA (ACCCC) binds to the TGGGG at the 3’ protruding end of the lagging strand template

2. Telomerase template RNA (AACCCC) serves as a template for the extension of the 3’ end of the lagging strand template with the addition of an extra TTGGGG

3. This is repeated several times

4. The extended 3’ end of the lagging strand template is used as a template to fill the 5’ gap in the lagging strand

Central Dogma of Molecular Genetics

DNA → Transcription → mRNA, rRNA, or tRNA → Ribosome → Translation → Protein

Cellular RNA Types

Generated by transcription

In all cells:

mRNA → protein

tRNA: Transfer RNA

rRNA: Ribosomale RNA

Eukaryotes have many types exclusive to them

Only in Prokaryotes: CRISPR RNA (crRNA): a precursor

A Transcription Unit

A segment of DNA that encodes an RNA molecule

Consists of: a promoter, the RNA-coding region (the gene), and a terminator

The gene is only the transcribed portions of the transcription unit

Transcription

The transfer of genetic material from DNA by the synthesis of a complementary RNA molecule

Requirements: DNA, RNA polymerase, free NTPs, no primers

Initiation of RNA Synthesis

NTP + NTP → NTP-NMP + PP

The initial nucleotide of the chain (5’) remains as a nucleoside triphosphate

Elongation of RNA Synthesis

NTP-(NMP)_n + NTP → NTP-(NMP)_n+1 + PP

Antisense Strand

The transcribed/template strand

Because it needs to be antiparallel

Sense Strand

The nontemplate strand

Transcription in Bacteria

Catalyzed by RNA polymerase

The Promoter: the binding site for RNA polymerase on the DNA molecule, located right before +1(transcription initiation site), and is not transcribed

The sigma factor recognizes the promoter and directs RNA polymerase to it; dissociates after initiation

DNA double helix is unwound and denatured locally

Transcription continues at 50 nucleotides/sec until it hits the terminator sequence

Termination of Transcription in Bacteria

Depends on the formation of a hairpin loop at the terminator site

Forms after inverted repeats in the gene are transcribed into the RNA molecule and form intramolecular base pairs

Formation destabilizes the DNA-RNA pairing and transcription ends

Rho-Dependent Termination

The Rho protein, a helicase, is needed to break the DNA-RNA pairing

Termination in Eukaryotes

Requires the activity of Rat1 exonuclease

After the protein-coding region is transcribed, the RNA molecule is cleaved by RNA endonuclease

Then Rat1, a 5’ to 3’ RNA exonuclease, binds to the unprotected 5’ ends of the trailing, non-coding RNA fragmentt and begins to degrade it, until it catches up with RNA polymerase

The complete degradation of the trailing piece terminates transcription

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

DNA arrays consisting of a number of short palindromic sequences that are separated by spacer sequences

Spacers are derived from invading DNA molecules, such as bacteriophages; the basis for adaptive immunity

CRISPR-CAS System

1. Acquisition: foreign dsDNA enters the cell and is identified and processed. A piece of it is inserted into the CRISPR array as a new spacer

2. Expression: the entire array is transcribd into a long CRISPR precursor RNA, then cleaved by a CAS (crispr associated) protein into CRISPR RNAs (crRNA), each one containing one spacer homologous to a foreign DNA. Each crRNA combines with a CAS protein to form an effector complex

3. Interference: if the same foreign DNA enters the cell again, the effector complex can quickly recognize it and the CAS protein cleaves it

Colinearity

Suggests that the number of nucleotides in a gene should be proportional to the number of nucleotides in the mRNA and to the predicted number of amino acids in the protein encoded by that gene

True for bacterial and viral genes

Rarely true for eukaryotic genes because their structure is different (the genes are interrupted)

Exons

The translated sequences of a gene

Introns

DNA sequences within a gene and between exons

They are transcribed, but don’t appear in the final RNA product

Non-coding

Pre-mRNA in Eukaryotes

After transcription, the primary mRNA transcript (pre mRNA) is processed to remove introns via RNA splicing

Mature mRNA

Contains only the exons and the 5’ and 3’ untranslated regions (UTRs)

Group 1 Introns

Have the enzymatic activity for self-splicing: they can remove themselves

Present in many precursor RNAs in prokaryotes and eukaryotes, but not in humans

Alternative Splicing

pre-mRNA can be processed in more than one way, so that one gene can generate more than one product

Alternative Splicing of the Preprotachykinin in mRNA

The gene contains 7 exons that are all transcribed into a pre-mRNA, but the pre-mRNA is spliced according to the tissue where the gene is expressed

β-preprotachykinin (substance K): found in the thyroid, intestines, and nervous system; mature mRNA contains all 7 exons

α-preprotachykinin (substance P): found only in the nervous system; mature mRNA lacks exon 6, which

codes for the K domain

Shine-Delgarno Sequence

Only present in prokaryotes

The ribosome-binding site during translation

The Methylguanosine Cap

Only in eukaryotes

A methylguanosine nucleotide plus several methyl groups are added to the 5' end of the mRNA

Protects the end from degradation by exonucleases

Polyadeylation

After the removal of the terminator by an RNA nuclease, 200-250 adenosine (A) nucleotides are added to the 3’ end of the mRNA forming the poly(A) tail