Exam 1 Flashcards
What is the dependent variable?
What you are measuring/what you care about/the outcome/the effect
What is the independent variable?
What you are manipulating (in hopes of affecting the dependent variable)/ the potential cause
What is the negative control?
The independent variable will not affect the dependent variable, tells us baseline information about the dependent variable
What is the positive control?
The independent variable will affect the dependent variable in a measurable way
What is the experimental/treatment control?
Unknown outcome, what you are testing
What are standardized variables?
Things that are kept the same for treatment and control groups to isolate cause and effect
What is the central dogma of biology?
DNA makes RNA through transcription, RNA makes and amino acid chain through translation, and an amino acid chain makes a protein through folding
What are proteins?
Linear chains of amino acids which fold to make complex shapes capable of doing a specific task in the cell
What determines the shape and abilities of a protein?
The order of specific amino acids in a protein
Describe the structure of DNA
DNA is make of 2 strands of nucleotides, with the nucleotides within a singular strand linked together by covalent bonds, and with the stands being help together by hydrogen bonds between base pairs
What are the 4 different nucleotides with 4 different nitrogen bases?
Thymine, cytosine, adenine, and guanine
What is a nucleotide composed of?
A sugar, a phosphate group, and a nitrogen base
What differentiates nucleotides?
Their nitrogen base
What nitrogen bases are pyrimidines?
Thymine and cytosine
What nitrogen bases are purines?
Adenine and guanine
What is the difference between pyrimidines and purines?
Pyrimidines have one carbon ring and purines have two
Where on the nucleotide is the phosphate group attached?
The 5’ carbon
Where on the nucleotide is the hydroxyl group attached?
The 3’ carbon
What is a phosphodiester bond?
The bond between the 5’ phosphate group and the 3’ hydroxyl group
How are DNA strands made?
By attaching the 5’ phosphate to the 3’ hydroxyl, which creates and strand with a 5’-P end and a 3’-OH end
What did Edwin Chargaff discover?
He discovered that, for any organism’s DNA, the amount of adenine=the amount of thymine and the amount of guanine=the amount of cystosine
What did Rosalind Franklin contribute to the discovery of the structure of DNA?
She took a picture of DNA via x-ray crystallography
Which bases pair together?
Purines pair with pyrimidines (adenine with thymine and guanine with cystosine)
What are the key features of the DNA double helix?
- Anti parallel strands (one stand goes in the 5’ to 3’ direction and the other goes in the 3’ to 5’ direction)
- Sugar phosphate “backbones”
- Bases “glue” stands together with H-bonds
How many hydrogen bonds does the adenine thymine bond have?
2
How many hydrogen bonds does the guanine cytosine bond have?
3
Which base pairing has a stronger bond?
Guanine and cystosine
What is the common form of a DNA helix? What other forms are there?
The common form is B-DNA, but there is also A-DNA and Z-DNA
Which DNA helices are right handed?
A and B
Which DNA helices are left handed?
Z
What do base pairing angles create?
Major and minor groove
What does underwinding of DNA create?
Negative supercoils
What does over winding of DNA create
Positive supercoils
Which type of supercoil can assist in separating DNA strands?
Negative supercoils
What is a genome?
The full hereditary information for an organism (complete set of DNA)
What is a chromosome?
A large, continuous DNA molecule
What are the 2 classes of cells?
Eukaryotic cells and prokaryotic cells
What are the characteristics of prokaryotic cells?
- Domains: archaea and eubacteria
- Smaller (1-5 µM) than eukaryotic cells
- DNA stored in cytoplasm (no nucleus)
- Only single celled organisms
What are the characteristics of eukaryotic cells?
- Domain: eukarya
- DNA stored in nucleus (surrounded by membrane)
- Organelles (“little organs”) surrounded by membranes with specialized jobs
- Larger (10-100 µM) thank prokaryotic cells
- Single and multi-celled organisms
What are the characteristics of prokaryotic genomes?
- Diverse
- One or more chromosomes
- Chromosomes can be linear or circular
- Can have small, circular plasmids
What are the characteristics of eukaryotic genomes?
Multiple linear chromosomes
What are eukaryotic chromosomes made out of?
Chromatin
What is a nucleosome?
DNA molecules wrapped around histone proteins
What is the order of packing patterns from DNA to mitotic chromosme?
DNA to nucleosome to 30 nm filament to extended form of chromosome to condensed section of chromosome to mitotic chromosome
What proteins help pack DNA?
Cohesion proteins
What happens when chromosomes are condensed?
When DNA is more tightly packed, the genes are less likely to be read to make proteins
What is euchromatin?
Loosely packed DNA actively being read to produce proteins
What is heterochromatin?
Tightly packed DNA not being read
What loosens DNA packing?
Acetylation of histone proteins
What tightens DNA packing?
Methylation of histone proteins
Describe the structure of RNA compared to DNA
- RNA is single stranded rather than double stranded
- RNA has uracil instead of thymine
- RNA has a different sugar than DNA (ribose instead of deoxyribose)
What can RNA base pair with?
- Nitrogen bases on the same RNA molecule
- Nitrogen bases on different RNA molecules
- Nitrogen bases on DNA molecules
What is the folding of RNA into 3D structures based on?
Base pairing
What is the origin of replication?
Specific sites (sequences) where DNA replication starts
How many origins of replication does E. coli have?
1
How many origins of replication do eukaryotes have?
Many (faster for larger genomes)
What is the replication bubble?
Expanded area of replicated DNA
What is the replication fork?
Site of active replication
How many replication forks are there per replication bubble?
2
What is the function of DNA polymerase?
Adding new nucleotide to the 3’ end of a DNA strand
Where does the energy to add a new nucleotide to a strand of DNA come from?
New nucleotides come in as nucleotide triphosphates (like ATP), the loss of two phosphate groups releases energy to power the connection of the nucleotide to the strand
What is the function of DNA helicase?
Unwinds double-stranded DNA, but overwinds the DNA in front of it, which requires topoisomerase to reduce strain
What is the function of primase?
Builds short RNA strands called primers that DNA polymerase can work from to build a DNA strand
How does primase work?
- Primase binds to the template strand and synthesizes an RNA primer
- When primer is complete, primase is released and DNA polymerase binds and synthesizes new DNA
In which strand does replication occur continuously?
Leading strand
In which strand does replication occur discontinuously?
Lagging strand
In which strand is DNA synthesized towards the replication fork?
Leading strand
In which strand is DNA synthesized away from the replication fork?
Laggin strand
What are Oakazaki fragments?
A series of segments of DNA that are synthesized on the lagging strand
What happens to the RNA primers on the lagging strand?
They are replaced with DNA by DNA polymerase
What is the function of single stranded binding proteins (SSPBs)
They prevent lagging strand from folding on itself and blocking replication
What are the specific functions of DNA polymerase III (main DNA polymerase)?
- 5’ to 3’ polymerase
- 3’ to 5’ exonuclease (for proofreading)
What are the specific functions of DNA polymerase I (specialized DNA polymerase)?
- 5’ to 3’ polymerase
- 3’ to 5’ exonuclease (for proofreading)
- 5’ to 3’ exonuclease (for replacing RNA primers)
What is the function of an exonuclease?
Cuts nucleotides off end of nucleic acid strand
What is the function of DNA ligase?
Connects adjacent strands of DNA together to combine Okazaki fragments to form one continuous new strand
Why does only adding nucleotides to the 3’ end of a strand allow for proofreading and removal of incorrect nucleotides?
Adding nucleotides to the 5’ end if one nucleotide is removed by proofreading does not require the triphosphate bond to be cleaved, providing no energy for the bond, while adding nucleotides to the 3’ end if one nucleotide is removed by proofreading requires the triphosphate bond to be cleaved, providing energy for polymerization (bond made)
What is the end replication problem?
Eukaryote’s linear chromosomes can never be fully replicated due to lagging strand dynamics, chromosomes shorten during each replicative cycle
What is a telomere?
Non-protein coding repetitive sequence found at the ends of chromosomes
What is the function of telomeres?
They act as a buffer to protect protein coding genes, as they shorten with every replication cycle (part of DNA that shortens instead of protein coding DNA)
What is limited by telomeres?
Replicative potential of cells (this prevents cancer, but may contribute to aging)
What is telomerase?
Protein-RNA complex (RNA- directed, DNA synthesis)
What is the function of telomerase?
It maintains telomere length in gamete producing cells
How does telomerase work?
- Telomerase extends the 3’ end of a DNA strand
- The other strand is extended the usual way by primase, DNA polymerase, and ligase
What is polymerase chain reaction (PCR)?
In vitro method for making copies of DNA sequences
What are the uses of PCR?
Molecular cloning, diagnostics, forensics, DNA sequencing, gene expression analysis, etc
Who invented PCR? What was his goal and solution
Kary Mullis
Goal: make more of the DNA he was studying
Solution: use biochemistry of DNA replication, but use heat to break DNA h-bonds and employ short DNA “primers” to define the amplification region
What initiates replication in PCR?
PCR primers
What are PCR primers?
Short DNA sequence (oligonucleotides, usually 15-20 nucleotides) produced synthetically and added to PCR reaction
What are the components of PCR?
Template (source) DNA, oligonucleotide primers, DNA polymerase, DNA nucleotides (dNTPs), buffers/salts
What is the process of PCR?
- Denature (95ºC): split DNA strands (break H-bonds with heat
- Anneal/prime (50-60ºC): reduce temp, sequence- specific primers bind (anneal) to target DNA (primers define amplification region (amplicon))
- Extend (72ºC): DNA polymerase extends from primer using target as a template
STEPS ARE REPEATED
What organism’s polymerase is used for PCR and why?
The DNA polymerase from Thermus aquaticus (Taq), a bacteria found in the hot springs in Yellowstone, is used because its DNA polymerase will not denature during the heating of DNA to separate strands
Why does the primer sequence influence annealing temperature?
GC base pairs are stronger than AT base pairs, so they can anneal at higher temperatures, also longer sequences can anneal at higher temperatures
How is Sanger sequencing different from PCR?
- Only one primer is used
- No chain reaction
- Dideoxynucleotides (ddNTPs) are included to prematurely terminate strand synthesis
What is the function of dideoxynucleotides (ddNTPs)?
They stop strand synthesis
What is gel electrophoresis?
Uses electricity to separate DNA molcules by size(number of nucleotides)
What is the process of Sanger sequencing?
- PCR with fluorscent, chain-terminating ddNTPs
- Size separation by capillary gel electrophoresis
- Laser excitation & detection by sequencing machine
How many nucleotides can Sanger sequencing sequence?
100s-1000s of nucleotides in one read
What is a contig?
A continuous segment of sequence created through overlapping “reads” (sequences)
What is coverage?
Number of copies of genome sequenced, more coverage is needed for new genome assembly
What do reference genomes allow for?
New individual genome sequences with less coverage
What is the function of RNA polymerase?
Separates the strands of a DNA molecule to build a new RNA molecule 5’ to 3’ that is complementary to one of the DNA strands
What do promoter DNA sequences mark?
The beginning of genes
What is the function of eukaryotic general transcription factors?
They bind promoters and recruit RNA polymerase
What is TBP?
TATA box binding protein
What is Rho-indpendent (intrinsic) termination of transcription?
Termination cause by hairpin in RNA transcript
What is Rho-dependent termination of transcription?
Uses Rho translocase to remove RNA polymerase and DNA from the transcript
How is RNA modified after transcription?
A 5’ methyl-G cap and a 3’ poly-A tail are added
What is the purpose of the 5’ methyl-G cap and 3’ poly-A tail?
Facilitate export from the nucleus, protect from degradation, facilitate translation (ribosome recruitment)
What are exons?
Protein coding mRNA
What are introns?
Non-protein coding mRNA
What are removed from mRNA before exit from nucleus?
Introns
What is RNA splicing?
The process of removing introns and connecting exons before the mRNA exits the nucleus
What is a spliceosome?
Protein-RNA complex that carries out RNA splicing, contains snRNPs
What are snRNPs (“snurps”)
Small nuclear ribonucleoproteins, contains snRNA (small nuclear RNA) that targets complex based on intron sequences
How is mRNA read during translation?
5’ to 3’ direction to build new proteins in the N-terminus to C-terminus direction
What is a codon?
A set of 3 RNA nucelotides
What do codons correspond to?
One amino acid
How is the genetic code redundant?
multiple codons per amino acid
How is the genetic code unambiguous?
only one amino acid per codon
What is covalently bonded to tRNA?
An amino acid
What is an anti codon?
Attached to tRNA to base pair with the mRNA codon
What is the function of amino-acyl tRNA synthetase?
Attaches appropriate amino acid to tRNA
What are the components of the ribosome?
-rRNA protein complex
-2 subunits come together to initiate translation
-3 tRNA binding sites
What is the site of translation?
Ribosomes
What amino acid do all polypeptides begin with?
Methionine (start codon AUG)
Describe ribosome recognition and binding of the 5’ end of mRNA in bacteria
mRNA ribosome binding site (Shine-Delgarno Sequence) base pairs with rRNA from small subunit, lining up correct AUG start
Describe ribosome recognition and binding of the 5’ end of mRNA in eukaryotes
-Small ribosomal subunit binds 5’ cap of mRNA
- Kozak sequence helps ribosome find AUG
What defined the reading frame of translation?
Initiation of translation/position of start codon
What are the steps of translation?
- New tRNA with correct anti codon binds to next mRNA codon in A site
- Growing amino acid chain is connected to amino acid from new tRNA
- Old tRNA leaves and message slides over to put tRNA holding peptide in P site and to make for for next tRNA in A site
What is the function of stop codons?
To recruit protein factos that terminate translation
What is termination (of translation)?
Stop codon recruits release factor (protein, not tRNA), translation complex is disassembled
What are polyribosomes (polysomes)?
Multiple ribosomes simultaneously translate the same mRNA
In what type of cell can transcription and translation occur at the same time and why?
Prokaryotic cells because the genes don’t have introns and the cells don’t have a nucleus, so transcription and translation can happen at the same time
What do open reading frames (ORFs) contain?
A start codon and no premature stop codons
What do ORFs help to predict?
Gene locations, especially in prokaryotes
Why are ORFs less useful for gene predictions in eukaryotic genomes?
Removal of introns can remove stops or change reading frame
What is a substitution mutation?
One nucleotide is replaced by another
What is a deletion mutation?
One or more nucleotides are removed from the gene
What is an insertion mutation?
One or more nucleotides are added to a gene
Are mutations bad?
Sometime, mutations can be good, bad, or neutral
What are the types of substitution mutations?
Missense, silent, and nonsense
What is a missense mutation?
Mutation that causes the wrong amino acid
What is a example of something caused by a missense mutation?
Sickle cell disease
What is a silent mutation?
Mutation that causes the same amino acid
What is a nonsense mutation?
Mutation that creates a stop codon
What are single nucleotide polymorphisms (SNPs) a result of?
Substitutions
What is a polymorphism?
Variation in DNA sequence
What is an SNP
Individual nucleotide difference tween different people
What types of mutations are frameshift mutations?
Deletions and insertions
What is a frameshift mutation?
Mutations that cause a different set of codons to be read after the mutation (a different reading frame)
What types of mutations cause more drastic protein changes?
Deletions and insertions
What determines cell type and what a cell can do?
The active proteins present in a cell
What is gene expression?
The transcription and translation of a gene to make an active protein
Do all cell types have the same DNA?
Yes
What is the most common way to control a protein’s presence?
Regulation of when a gene is transcribed
What is constitutive expression?
When the gene the is expressed at all times
What is an inducible expression?
When gene expression is off until actively turned on
What is repressible expressions
When gene expression is on until actively turned off`
How can activator transcription factors increase gene expression?
They can bind enhancer DNA, which increases gene expression by bringing general transcription factors
What are trans DNA sequences?
Anywhere genes for transcription factor proteins
- Activators= increase transcription
- Repressors= decrease transcription
What are cis DNA sequences?
Near gene binding sites for transcription factors
- Enhancer= attracts activator
- Silencer= attracts repressor
What is an operon?
Multiple genes with one promoter?
Do operons exist in prokaryotes or eukaryotes?
Prokaryotes
What are the enzymes required to import and break down lactose?
β-Galactosidase and galactoside permease
When E. coli is growing in a medium with glucose and lactose, what does it eat first?
The E. coli eats glucose first, then lactose
What must be present for the lac operon to be on?
Lactose
What must be absent for the lac operon to be on?
Glucose
What is the positive control of the lac operon?
Catabolite activating protein (CAP) transcription factor
What is the negative control of the lac operon?
Lac repressor transcription factor
What happens when there is no lactose present?
The repressor, which senses lactose, blocks the promoter when it is binded to the operator of the genes for lactose enzymes, so RNA polymerase cannot attach to the promoter and transcription and gene expression is blocked
What happens then lactose is present?
The repressor senses the lactose and releases from the operator, which allows RNA polymerase to bind to the promoter and transcription and expression of the genes for the lactose enzymes
When is cAMP present?
When glucose is low
What occurs when cAMP is absent?
CAP does not bind to the promoter, so transcription occurs at a low rate
What occurs when cAMP is present?
CAP binds to the promoter and increases RNA polymerase activity
What causes CAP to bind to the promoter?
Low or absent glucose, which causes cAMP to be produced, which allows CAP to bind to the promoter and transcription and RNA polymerase activity to occur
What does strong expression of the lac operon require?
Release of repression and transcriptional activity (high lactose and low glucose)
What is the cis regulatory sequence for the lac operon?
Binding site for transcription factor
What is the rans regulatory sequence for the lac operon?
Gene for transcription factor
What can be caused by mutations to regulatory DNA sequences?
Genetic diseases and cancer by changing the availability of certain proteins in cells
What does alternative splicing do?
Creates different protein variants in different cells or at different times
How does alternative splicing aid in neural connections?
Provides a signature for each neuron, allowing neurons to correctly “wire up”
What does Dscam regulation?
Neuron adhesion
How many alternatively spliced forms does Dscam have?
19,008
What helps to start translation?
Eukaryotic initiation factors (eIFs)
What happens when eIF2 is phosphorylated?
Translation is blocked
What happens when eIF2 is not phosphorylated?
Translation occurs
Why do some viral mRNAs use internal ribosome entry site (IRES)?
To circumvent initiation factors
What are the untranslated regions?
The mRNA before the start codon and after the stop codon?
What is the function of untranslated regions?
They regulate mRNAs
How does RNAi regulate mRNA?
Highly specific to one mRNA
How does miRNAs regulate mRNA?
Regulates multiple mRNAs
What is the function of topoisomerase?
To reduce strain of overwinding
