Unit 4 Flashcards
What is DNA made of?
genes
DNA -> RNA
transcription
RNA -> Protien
translation
Protein express into
specific phenotypes (every gene produces a specific protein)CH
Chromosomes
compressed DNA/Proteins
Properties of Genetic Material
- carries information
- capable of being copied
- capable of undergoing change
what biological marcomolecule contained genetic info?
DNA
Griffiths Experiment
- found what macromolecule contained genetic info with mice (DNA)
- when nonpathogenic dead cells and nonpathogenic living cells come together what makes this kill the mice?
- figured out the cells are transformed
Avery, MacLoed and McCarty Experiments
- wanted to see what made it transform
- when the R cells and S cells were together they were then introduced to protease (degrades protein) and DNase (degrades DNA) to see what makes it transform
- only ones given protease died meaning DNA is REQUIRED for transformation
Bacteriophage T2
also test to make sure DNA was needed for transformation
We can track structure with radioactive isotopes
- like S35, radioactive so it releases energy and decays over time allowing us to track structure
Hershey and Chase
- had one protein coated and one DNA coated phage, infected bacteria with it and only the bacteria coated carried on showing DNA was genetic material
Nucleotides Structure
- phosphate group (binds to C5)
- ribose group (In RNA), deoxyribose group (in DNA, in carbon 5 the oxygen is gone)
- nitrogenous base (purine or pyrimidines)
- AMP = 1 phosphate
- ADP = 2 phosphate
- ATP = 3 Phosphate
Pyrimidines
- 1 circle
- Cytosine, Thymine, Uracil
Purines
- 2 circles
- adenine and guanine
Chargaff
%A = %T, and %G = %C
can be in different percentages tho like 70:30 etc, but equal 100
DNA Structure
- double helix
- comes from C5 to C3, opposite side comes from C3 to C5 with phosphodiester bond
- A-T (2) and C-G (3) have hydrogen bond
- sugar backbone
Base Pairing
one strand of DNA holds info for other strand and vice versa, DNA order carries info that’s reflected in our phenotypes that make us different
Terminator Nucleotides
- have no available C3 so DNA chain is terminated if one of these is added because it has a OH bonded to it
Gel Electrophase
- DNA is negative, goes to bottom which is positive by particle size
DNA Packaging
- DNA is wrapped around histone proteins (8 proteins in 1 histone, a cube shape.
- DNA wraps around twice
- histones are wrapped into a chromatin fiber where it further condenses into the chromosome
Nucleosome
DNA wrapped around histone 2x
DNA Sequencing
- DIRECTIONAL
- different sequences mean different info
How is DNA copied?
- Semi-Conservative
Why E Coli is used to study DNA replicaiton
- easy to use
Meselson-Stahl Experiment
15N (radioactive) had RED, 14N (normal) had orange, as generations kept going on, amount of red got smaller but NEVER disappeared so DNA was semi-conservative
How DNA is replicated
- strands seperate
- new strand grows from 5C, new bases are added from parent bases as template
How DNA helix is unwinded?
- Origin of Replication (many than expand to seperate)
Helicases
enzymes that untwist the double helix at replication forks
Single-Stranded Binding Proteins
- bind to and stabilize single-stranded DNA
Topoisomerase
- corrects overwinding ahead of replication forks by breaking, swiveling, and rejoining DNA strands
Primer
starter of a new DNA strand (temp)
How new DNA is polymerized?
- DNA polymerase builds DNA in 5C -> 3C direction
- it creates a phosphodiester bond between 3C OH of last nucleotide and the 5C of phosphate of incoming.
DNA Polymerase
- creates covalent bonds between 3C of OH and 5C of Phosphate of next
- all DNA polymerase needs a free 3C OH to make new DNA
Where does 3C of OH come from?
RNA Primer
Primase
- makes RNA using DNA as template
Primer
short strand of DNA for DNA polymerase to created new strand of DNA
Leading Strand
- made continously
Lagging Strand
- made discontinuously
- built in small fragments called the Okazaki Fragments
DNA Ligase
created phosphodiester bond between 3 OH and 5 phospjate (the gaps inbetween)
Replication bubble
- each has lagging/leading
Error in Replication?
- happens but can usually be repaired in the next replication
- DNA Polymerase can also proofread, as it checks DNA is checked for incorrect bases and can then pause and fix mistake before moving on (removes it and replaces)
Post-Replication Repaire
- 2 restriction enzymes come in and get ride of that segment of misincorporation and completely replace it
- error rate after is low BUT NOT ZERO, sequence changes can be permanent and passed on, these mutations are sources of genetic variation
Replicating Ends of Linear Chromosomes
- Telomers help by creating extensions so it doesnt shrink
Telomerase
- created long repeates called telomeres on ends of linear chromosomes
- not active in most human cells
- examples of reverse transcriptase
Reverse Transcriptase
used RNA as a template for DNA synthesis
Transcription
the synthesis of RNA using information in the DNA (transcribing it so the message can be used in nucleic acid language)
Translation
synthesis of polypeptide using info in the mRNA (translating the message from nucleic acids to amino acids)M
Genetic Code
- 300 total AA, only 20 in humans)
- three letters, (UCAG, UCAG, UCAG) are side in chart
- codons read 5C to 3C
- codon is degenerate, AA can have a form of many different codons
Special Codons
- AUG = start
- UAA, UAG, UGA = stop
Transcription Steps
- Initiation
- Elongation
- Termination
Structure of Gene
- Promoter (start)
- region that is transcribed in the middle (copied into RNA)
- terminator (end)
Promoter
- initiation of transcription
- DNA polymerase binds to the promoter in prokaryotes, in eukaryotes it’s recruited to promoter
DNA Polymerase Needs what to Start Transcription?
- Needs -35 Region
- Needs -10 Region
- won’t detect promoter unless these are here
Elongation
- transcription bubble
- DNA polymerase synthesized mRNA in 5 -> 3
- template strand reads 3 -> 5
mRNA processing only in eukaryotes
- pre-mRNA undergoes 3C Poly-A tail, 5C cap, and splicing
- resulting mRNA leaves nucleus and is translated by ribosomes
Poly-A Tail and 5C Cap
- facilitate the export of mRNA to the cytoplasm
- protect mRNA from hydrolytic enzymes
- help ribosomes attack to 5C end
Introns
long noncoding stretches of nucleotides between coding regions
Splicing
- many eukaryotic genes have introns, splicing removes introns and joins exons creating a molecule that codes continuously
Exon
coding region
How are codons interpreted?
a tRNA with an amino acid on the 3C, has an end with 3 anticodons that bind to the mRNA
tRNA
go into cytoplasm, grab AA, then put them into a sequence on the mRNA
Anticodon pairing showing “wobble”
3rd letter of the codon most time doesn’t match up, so it wobbles
Ribosomes
large units of enzymes
- WHAT? facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis
- contains both RNA and proteins
- two ribosomal subunits (large and small)
Large Subunit
- E site, P Site, A Site, Exit Tunnel
Small Subunit
- mRNA binding site
A Site (binding site)
the amino acid comes in here and is held until it’s needed
P Site (binding site)
AA moves to P site where it binds with the incoming mRNA
E Site (exit site)
- moves to E where it then exits
mRNA Binding Site
- holds the incoming mRNA
Steps of Translation
- Initiation
- Elongation
- Terminantion
Translation Initiation
- Initiator tRNA (with the amino acid) binds to the mRNA sequence on small ribosomal subunit and anticodons are paired
- GTP energy is used and GDP is released because it needs energy to recruit the large ribosomal subunit
- large joins on and initator tRNA is in the P Site
Elongation Translation
- Codon recognition and another tRNA joins on in A site (energy used)
- amino acid chain from previous tRNA joins onto new tRNA in the peptide bond formation
- then translocates where old tRNA is released in E Site using energy and new one moves into the P Site
Termination Translation
- stop codon is attached to mRNA, so the ribosome reads the stop codon and stops the process
- release factor promotes hydrolysis were old tRNA is released again but stop codon doesn’t move to P Site and the amino acid chain is released
- then the ribosomal subunits and other components detach
Polyribosomes
- many ribosomes on one mRNA making lots of proteins
Translation in Eukaryotes
- Free Ribosomes (proteins stay in the cytoplasm)
- Bound Ribosomes (on the ER, integral membrane proteins, and are secreted out of the cell too)
- translation is initiated on free ribosomes in the cytosol, ribosomes can then be docked into the rough ER if the protein needs to enter the endomembrane system
Mutation
heritable change in the genetic material
- in single celled, they are passed to the next generation
- in multicelluar organism only the sex cell mutations can pass these one
Causes of Mutations
- Spontaneous (errors in DNA replication or repair, chemical changes in DNA)
- Induced (result from damage to the DNA and failure to repair)
Point Mutations
- Base Substitutions (total number of bases stay the same, but a single base pair is altered)
- Insertions or Deletions (total number of bases changes)
Differential Expression of Genes
- prokaryotes and eukaryotes regulate gene expression in response to environmental changes
- in multicellular organisms gene expression regulated development and is responsible for differences in cell types
Gene Expression in Prokaryotes
- often respond to environmental change by regulating transcription
- use the operon model
Operator
a segment of DNA that controls functionally related genes like an on-and-off switch
Operon
ENTIRE stretch of DNAS that includes the operon, promoter, and the genes they control
- they all work together
Trp Operon
- in E coli. the genes are located right next to each other in the trp operon
- these five are only needed when the cell needs to make the tryptophan (it’s anabolic cause it builds up)
- when tryptophan is plentiful it acts as a co-repressor binding to a repressor protein to turn OFF the operon
- when tryptophan is absent, repression protein is inactive and does not bind to the operator so genes ARE transcribed
Co-Repressor
- attached to a repressor protein to STOP the transcription of genes
- when gone the repressor doesn’t attach to the operon
lac Operon
- the 3 genes needed to break down lactose in E coli. are right next to eachother in lac operon
- these are only needed when the cell needs to break down lactose for energy (catabolic cause breaking down)
- when lactose is plentiful it acts as an inducer and binds to the repressor protein to inactivate it, turning ON the operon
- when lactose is absent the repressor protein is active and binds to the operator sequence, turning OFF the operon
Inducer
- stops repressor protein from stopping the operon
Positive Gene Regulation in Prokaryotes
- some operons are also subject to positive control through a stimulatory protein called an activator
- CAP is an activator, in the absence of cAMP CAP does not bind to the promoter but transcription still occurs just less, when cAMP IS there CAP binds to promoter and increases polymerase acitivty
CAP regulation of lac operon
- glucose preferred food of E coli.
- in absence of glucose, cAMP concentrations increase, activating CAP, which binds to promoter of lac operon so it goes quicker
- in presence of glucose cAMP concentrations are low, CAP can not be activated and will not bind to promoter which means operon is slower
To sum up lac operon…
- turned on in presence of glucose
- when glucose is scarce the activator CAP is sent to the lac operon to increase rate of transcription
Activator
increases the rate of transcription of an operon so it’s a positive regulator
- specific transcription factor
Regulation of Gene Expression in Eukaryotes
- chromatic (epigenetic)
- transcription
- post-transcription
- translation
- post-translation
- all have roles in creating the perfect gene expression, and all have factors where at each level we can stop gene expression
Regulation of Chromatic Structure (Eukary)
- genes with highly packed heterochromatic are usually not expressed
- loosely packed euchromatic is associated with active gene expression
- chemical modifications to histones and DNA of chromatin influence both chromatic structure and gene expression
Histone Modifications in Chromatin Structures (Eukary)
- When a methyl group is bound to the histone it wraps them up tighter, causing genes to not be expressed
- when a acetyl group is bonded to histones is loosens the wraps and allows for genes to be expressed
DNA Methylation
- addition of methyl groups to certain bases in DNA is associated with reduced transcription, can also cause long-term inactivation of genes in cellular differentiation
Epigenetic Inheritance
- although chromatic modifications don’t alter DNA sequencing, they can be passed to future generations of cells
- this is called this its the inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence
Regulation of Transcription (Eukary)
- to initiate transcription, eukaryotic RNA polymerase requires the assistance of transcription factors
- general transcription factors are essential for the transcription of all protein-coding genes and bind to the promoter
- high levels of transcription of particular genes depend on control elements interacting with specific transcription factors which bind to enhancer elements
Transcription Factors
starting, elongation, etc
Enhancer Element
accelerating gene expression, suppressing gene expression
Specific Transcription Factors
can bind to enhancer elements OR repressors
DNA Bending Protein
helps fold DNA to make enhancer closer to the promoter/target gene
Enhancer
controls the process and needs to be close to the promoter
- a repressor can also bind to the enhancer region which prevents transcription
Transcription Re-Programming
- with the right transcription factors the cells can be reprogrammed into primary stem cells, then differentiated into body cells, then differentiated again into different stem cells to replace other damaged ones
Post-Transcriptional Gene Regulation (Eukary)
- Alternative Splicing
- mRNA stability
Alternative Splicing
- pre-mRNA is spliced to cut out introns
- the pre-mRNA can be alternatively spliced to create different proteins
- different kinds (look at slides)
RNA Binding Proteins
- increases our stabiltiy
- every RNA molecule has a defined lifespan and decays at a specific rate, the presence of RNA-binding proteins at the 5C or 3C UTR influences the stability either increasing this lifespan or decreasing based off our needs
Control of mRNA stability (miRNA’s)
- Negatively Regulate Gene Expression by;
- blocking translation of the mRNA
- degrading the mRNA (if bases are completely complentary)
miRNA (microRNA)
small single-stranded RNA molecules that can bind to the mRNA
Translation Gene Regulation (Eukary)
- translation of all mRNA’s in a cell may be regulated simultaneously
- miRNA can block translation (when eIF-2 is phosphorylated translation is blocked)
Post-Translational Gene Regulation
- many proteins aren’t activated until they are modified
- another way to control gene expression is to alter longevity of the protein
- when ubiquitin is in the presence of ATP it binds to a protein, this then goes into a proteasome where the ubiquitin is released, ADP is released and the protein is broken down into amino acids
