Genetics Exam 2

Central Dogma

1. DNA (DNA replication) - information is stored here

2. Transcription converts DNA into RNA - information is used by transcribing DNA to make RNA

3. Translation converts RNA into a protein

Eukaryotic DNA

Linear and organized into chromosomes; Tertiary structure (complex packaging of DNA - chromatin); Hierarchical folding of chromosome; Contains nucleosome (DNA wrapped around histone proteins)

DNA Structure

Two complementary and antiparallel nucleotide strands form a double helix

DNA Secondary Structure

Stable 3D structure; Watson and Crick’s helical structure

DNA Primary Structure

String of nucleotides joined together by phosphodiester linkages

The Human Genome is _____ Base Pairs in Length

3.2 billion

Nucleic Acids

Linear molecules made up of repeating subunits called nucleotides

Components of Nucleotides

Contain a nitrogenous base (purines and pyrimidines), phosphate group (negative charge), and either Ribose (RNA) or Deoxyribose (DNA)

Nucleotides are covalently linked together by ____

Phosphodiester Bonds (between 3’ OH and the phosphate group)

Deoxyribose

OH group on the 3’ carbon; Phosphate group on the 5’ carbon

Ribose

OH group on the 2’ and 3’ carbon; Phosphate group on the 5’ carbon

Adenosine Nucleoside

Ribose + Nitrogenous Base (no phosphate)

Adenosine Monophosphate (Nucleotide)

One phosphate group, Ribose, Nitrogenous Base

Adenosine Diphosphate (Nucleotide)

Two phosphates, Ribose, Nitrogenous Base

Adenosine Triphosphate (Nucleotide)

Three phosphate groups, Ribose, Nitrogenous Base

Nucleotides are linked in ____

Strands

dsDNA

Double stranded DNA; Two strands are not identical but are complimentary (A - T, G - C)

Complementary base pairs interact/bind to each other through ____

Hydrogen bonds; Hydrogen bonds are low energy and stabilize the molecule, but can also be disrupted by enzymes, or with the input of energy (ex. heat)

A and T have ___ hydrogen bonds between them

Two

G and C have ____ hydrogen bonds between them

Three

Antiparallel dsDNA

Polynucleotide strands run in opposite directions to each other; One side goes 5’ to 3’ while the other goes 3’ to 5’

One nucleotide is about ___ long

0.34nm

One complete turn is about ___ long

3.4nm

DNA’s double helix is about ___ wide

2nm

Two Types of Double Helix Models

Ball and stick model; Space-filling model

B DNA

Shape DNA takes in an aqueous environment; Double helix; What we know as DNA

Z DNA

Previously thought of as a rare form in desiccated organisms; Now seems to be in more places

4 Types of RNA Loops

Bulge loop, Internal loop, Multi-branched loop, Sternum loop

Structure of RNA Molecules

Almost always single-stranded (don’t have same stability as DNA); Finds complementary base pairing with itself to help stabilize it, which causes it to fold up, creating loops; Added hydroxyl group on every nucleotide (more reactive and unstable)

Nucleotides are synthesized in the ___ direction

5’ to 3’; Form bond between 3’ OH group and 5’ phosphate group and make a phosphodiester bond

Structure of a Transfer RNA

Many factors contribute to RNA’s tertiary structure; Base-pairing and base stacking within the RNA itself; Interactions with ions, small molecules, and large proteins

Genetic Material Criteria

1. Must contain information necessary to make an entire organism

2. Must be passed from parent to offspring (transmission)

3. Must be copied in order to be passed from parent to offspring (replication)

4. Must be capable of changes to account for known phenotypic variation in each species

In search of the genetic material, Griffith discovered the principle of ___

Transformation

Griffith

Isolated two strains of Strep. pneumoniae; One strain was virulent and had a polysaccharide coat that made colonies appear smooth; Virulent forms sometimes mutated into non-virulent forms and lost the coat, therefore appearing to be rough; When the Strep. was injected into mice, Rough / Non-virulent mice lived, and Smooth/Virulent mice died; In a mouse with heat that killed the smooth type, the mouse lived; In a mouse with heat that killed the smooth type mixed with rough type, the mouse died, and virulent bacteria was recovered in the autopsy

Avery, McCloud, and McCarty show ____ is DNA

Transforming principle; Showed that only DNA had the ability to transform

Hershey-Chase Experiment Basis

Confirmation that DNA is the genetic material; Bacteriophage T2 infects E.Coli and has a DNA genome with phosphorous, and a protein coat with sulfur;

Phage Life Cycle - Hershey and Chase

1. Phage attaches to E.Coli and injects its chromosome

2. Bacterial chromosome breaks down and phage chromosome replicates

3. Expression of phage genes produces phage structural components (proteins and DNA)

4. Progeny phage particles assemble

5. Bacterial wall lyses, releasing progeny phage

Isotope

Radioactive form of an element

Hershey-Chase Radioactive Labeling

Used this technique to track molecule of interest; Incorporated radioactive P in DNA and radioactive S in proteins

Hershey-Chase Experiment Findings

After centrifuging, 35S was found in empty viral coats, not bacterial cells, and new phages from cells had NO 35S; 32P was found in bacterial cells, not empty viral coats, and new phages WERE radioactive with 32P; DNA from infecting phages was passed on to progeny, NOT protein

Chargaff’s Rule

Found that the amounts of the 4 bases varied between species, but their ratios did not; A=T and C=G

Rosalind Franklin

Performed X-ray Diffraction on DNA Fibers; Diffraction pattern is interpreted using mathematical theory to provide information concerning the structure of a molecule

Watson and Crick

Discovered the 3D structure of DNA in 1953; Used existing data (including Franklin’s), molecular models, and knowledge of structural chemistry; Watson recognized an A could bond with a T and a G could bond with a C (this accounted for Chargaff’s rule)

Deoxyribonuleotide Triphosphates (dNTPs)

Essential building blocks and energy source for DNA

DNA Replication is ____

Semiconservative; Each strand of the DNA acts as a template to recreate the other strand through complementary base pairing

In DNA, one ____ is used to make two ____ of DNA

One double strand → Two identical strands

3 Models of DNA Replication

Conservative Model, Semiconservative Model (correct), and Dispersive Model

Conservative Model

Original strand acts as a template but an entirely new strand is made and the old strand remains intact; After the second round, the original strand is still used as a template, and the new strand is used as its own template to replicate (results in 1 old + 3 new)

Semiconservative Model

One strand is original and one is newly synthesized on both sides; After second round results in 2 half/half and 2 completely new

Dispersive Model

Old DNA would get broken up, act as a template to make new strands, and then would get “put back together”; Results in 4 half/half

In DNA Replication ____ is where we add a new nucleotide, and ____ is the building block

3’ OH, 3-phosphate group

Evidence that DNA is Semiconservative

1. Cells were first grown in media with heavy nitrogen (N15)

2. Cells were then grown in media with light nitrogen (N14)

3. Cells divided every 30 minutes

4. DNA was assessed with centrifugation to determine how much heavy and light nitrogen was in the DNA

5. There was one band of heavy nitrogen 15 and after letting bacteria grow (after 1 full found) all DNA was half heavy and half light because it was made with nitrogen 14 as well (half and half band)

6. After the second round, the half and half acted as a template, and light-only DNA started accumulating (equal amounts of half and half and just light bands)

Bacterial Chromosome Replication

Replication starts at the origin of replication; The replication fork is where the strands of DNA are separating; Replication occurs bidirectionally

DNA Helicase

Unwinds DNA and creates torsional strain

DNA Gyrase

Has topoisomerase activity; Called gyrase in bacteria and topoisomerase in eukaryotes; “Nips” the backbone and allows tension to unwind and reseals the backbone; Relieves torsional strain so DNA can continue replicating (without topoisomerase, DNA replication is stalled)

Single-Strand Binding Protein

Prevents DNA from re-annealing

DNA Ligase

Seals Okazaki fragments on the lagging strand and fixes breaks

Primase

Essential RNA polymerase enzyme that synthesizes short RNA primers on single-stranded DNA templates; Provides necessary 3’ OH group for DNA polymerase to initiate synthesis

DNA Polymerase can attach nucleotides only in the ___ direction

5’ to 3’

DNA Polymerase

Cannot initiate DNA synthesis by linking two nucleotides on a bare template strand; Needs a 3’OH (problem is overcome by RNA primers synthesized by primase); Two strands are anti-parallel and go in opposite directions (problem is overcome by synthesizing the new strands both toward and away from the replication fork)

Identifying Leading and Lagging Strands

Leading strand is synthesized towards the replication fork; Lagging strand is synthesized away from the replication fork

Primosome

Complex formed when DNA helicase and primase physically bind to each other; Physically associated with two DNA polymerase holoenzymes to form the replisome

DNA Polymerase Proofreading Function

Can identify a mismatched nucleotide and remove it from the daughter strand; Uses 3’ to 5’ exonuclease activity to digest the newly made strand until the mismatched nucleotide is removed; DNA synthesis then resumes in the 5’ to 3’ direction

Why is DNA replication fidelity high? (few mistakes are made)

1. Stability of base pairing

2. Structure of the DNA polymerase active site

3. Proofreading function of DNA polymerase

Replication of Eukaryotic Chromosomes

Similar to bacteria but more complex; Large linear chromosomes; Chromatin is tightly packed within nucleosomes; More complicated cell cycle regulation; Replication bubbles from multiple origins merge into completely replicated chromosomes

DNA Polymerases in Eukaryotes

Mammalian cells contain well over a dozen different DNA polymerases; Four have the primary function of replicating DNA (Alpha, Delta, Epsilon, and Gamma); Many are translesion-replicating polymerases

Translesion-Replicating Polymerases

Involved in the replication of damaged DNA; Can synthesize a complementary strand over the abnormal region

Alpha DNA Polymerase

Initiates DNA replication in conjunction with primase

Epsilon DNA Polymerase

Replication of the leading strand

Delta DNA Polymerase

Replication of the lagging strand

Gamma DNA Polymerase

Replication of mitochondrial DNA

Removal of an RNA Primer

Distinct from prokaryotic replication; Delta polymerase runs into a primer of adjacent Okazaki fragment and pushes a portion of primer into a short flap; Flap endonuclease then removes the primer; If the flap is too long, it is cleaved by DNA2 nuclease/helicase which cuts the long flap into a short flap; DNA ligase seals the two fragments

Telomeres

The complex of telomeric DNA sequences and bound proteins; Sequences consist of moderately repetitive tandem arrays (3’ overhang that is 12-16 nucleotides long); Typically consist of several G nucleotides and many T nucleotides; Linear eukaryotic chromosomes have telomeres at both ends

Replication Problem at Ends of Linear Chromosomes

At the 3’ ends of linear chromosomes, the end of the strand can’t be replicated because DNA Polymerases only synthesize DNA in the 5’ to 3’ direction and cannot initiate DNA synthesis on a bare DNA strand; The cell solves this problem by adding DNA sequences to the ends of telomeres through a mechanism catalyzed by telomerase

Telomerase Structure

Contains protein and RNA; The RNA is complementary to the DNA sequence found in the telomeric repeat, allowing the telomerase to bind to the 3’ overhang

Telomere Length in Cancer and Aging

Telomeres tend to shorten in actively dividing cells; Telomere DNA is about 8,000 base pairs at birth and can shorten to 1,500 base pairs in an elderly person; Cells become senescent when telomeres are short (lose their ability to divide); Insertion of highly active telomerase can block senescence; Cancer cells commonly carry mutations which increase activity of telomerase, which prevents telomere shortening and senescence (may be a target for anti-cancer drug treatments)

RNA Synthesis and Transport (steps)

All RNAs are synthesized in the nucleus; mRNA is exported to the cytoplasm and then translated into a protein

Central Dogma

Information is stored in DNA, that information is used when DNA is transcribed into RNA, and RNA is translated into a protein

Gene Expression

Using DNA to make a functional product (RNA or protein)

Regulatory Elements

Site for the binding of regulatory transcription factor proteins

Promoter

Site for RNA Polymerase binding; DNA sequences that “promote” gene expression; Direct the exact location for the initiation of transcription; Typically located just upstream of the site where transcription of a gene actually begins; Bases in a promoter sequence are numbered in relation to the transcriptional start site

Terminator

Signals the end of transcription

Codons

3-nucleotide sequences within the mRNA that specific particular amino acids

Template Strand

DNA strand actually transcribed/used as a template; Complementary to the RNA transcript

Coding Strand

Sense Strand/Non-template strand; Identical to RNA base sequence (except for the substitution of Uracil in RNA for Thymine in DNA)

Transcription - Initiation

Promoter functions as a recognition site for the transcription factors; The transcription factors enable RNA Polymerase to bind to the promoter; After binding, the DNA is denatured into an open transcription bubble

Transcription - Elongation/Synthesis of the RNA transcript

RNA polymerase slides along the DNA in an open complex to synthesize RNA

Transcription - Termination

A terminator is reached that causes RNA polymerase and the RNA transcript to dissociate from the DNA (in bacteria)

Are template strands all the same?

No; Each gene uses one DNA strand as a template, but the template strand used is not always the same

A ___ specifies the direction of transcription

Promoter; With regard to adjacent genes along a chromosome, some promoters direct transcription in one direction and others direct transcription in the opposite direction

-35 and -10 Sequences

Promoters in bacteria vary at the -35 and -10 sequences

Consensus Sequence

The most common promoter sequence; Likely to result in a high level of transcription; Sequences that deviate from the consensus sequence typically result in lower levels of transcription

Core Promoter

Relatively short; Typically consists of the TATA box (a Transcriptional Start Site) and one or more downstream promoter elements (DPEs); TATA box is not always present, but it is important in determining the precise start point for transcription

Enhancers

Usually 50-1000 base pairs and contain one or more regulatory elements; Can vary widely in their locations but are often found in the -50 to -100 region

Regulatory Transcription Factors

Bind to regulatory elements and influence the rate of transcription; Activators stimulate transcription and Repressors inhibit transcription

Proteins Needed for Transcription in Eukaryotes

1, RNA Polymerase 2 (catalyzes linkage of nucleotides in the 5’ to 3’ direction)

2. Six different proteins called General Transcription Factors (GTFs)

3. A protein complex called the mediator

Preinitiation Complex

Formed by the general transcription factors, mediator, and RNA polymerase 2, which assemble at the promoter of a gene (often containing a TATA box)

Without enhancers, the core promoter produces ___

Basal transcription (a very low level of transcription)

RNA Transcript Synthesis (Elongation)

RNA polymerase slides along the DNA, creating an open complex as it moves; RNA Polymerase slides along the template in a 3’ to 5’ direction, and synthesizes RNA in a 5’ to 3’ direction using nucleoside triphosphates as precursors

RNA Polymerase 1

Transcribes all rRNA genes (Except for the 5S rRNA)