Week 4 Flashcards
Why study DNA?
1) Essential for inheritance
2) Codes for proteins
3) Instructions for life processes
Why is DNA important?
1) Study genetics causes of disease
2) Design of gene therapies
3) Develop drugs
4) Forensic science
5) Genomic sequencing
6) Detect pathogens
7) Determine paternity
Human genome divided into:
# DNA molecules
# autosomes
# sex chromosomes
46 DNA molecules
22 autosomal pairs
2 sex chromosomes
DNA contains how many bases?
6 billion
DNA contains how many base pairs?
3 billion
How long would DNA be if unwound?
2 m long
Composition of histones
2 pairs H2A, H2B, H3, and H4
H1 resides outside as lock for DNA coiling around histone complex
Nucleosome
Histone complex with DNA wrapped 2.5 times around it; no H1 protein attached at this point
Polynucleosome
Histone complex with DNA wrapped 2.5 times around it; H1 protein attached and is tightly compacted on itself
H1 histone protein
Linker protein
Binds to entry/exit site of DNA
Needed to stabilize higher order chromatin structures
Chromatin
Collection of nucleosomes put together in a tight condensed area
Supercoiling of DNA
Allows DNA to compact even tighter around itself
Charge on histones
Positive
Charge on DNA
Negative due to phosphate groups
Euchromatin
Relaxed DNA
Transcriptionally active
Exposed to nuclease digestion
Heterochromatin
Condensed DNA
Transcriptionally LESS active
Protected from nuclease digestion
Role of topoisomerase
Change degree of supercoiling in DNA; essentially relaxes the supercoiling of DNA
Acetylation of Histones - does what?
Causes histones to become more neutral; will becomes looser and allow DNA to unwind
Transcription allowed to increase
Phosphorylation of Histones - does what?
Add negative charge
Will cause greater amount of steric hindrance between molecules - loosening DNA from histones
Transcription allowed to increase
Methylation of Cytosine - does what?
Compacts DNA even further than it was
Decreased transcription
What occurs during G1 phase of cell cycle
Protein synthesis increased
Which period of the cell cycle are cells more responsive to mitogenic GF’s and TGF-Beta?
G1
What percentage of genome is used to encode proteins?
1.5%
What percentage of genome is comprised of introns?
26%
What percentage of genome is Long Interspersed Elements (LINE)?
20%
Which LINE is most abundant (13% of genome)?
L1
What percentage of genome is Short Interspersed Elements (SINE)?
13%
Which SINE is the most abundant (7% of genome)?
Alu
What percentage of genome is comprised of transposons?
11%
What are transposons?
Moveable genes
Usually viral in origin
What percentage of genome is considered heterochromatin?
8%
What would be considered heterochromatin?
Centromeres and telomeres
What is a telomere?
Region of repetative nucleotide sequences associated with specialized proteins at end of chromosomes
What is a centrosome?
Protein that links pair of sister chromatids to one another during cell division
Types of issues with transposable regions of DNA?
1) Increase/decrease spacing between regulatory units = change expression
2) Insert/delete coding region for proteins = alter protein functions
3) Alter gene expression = formation of pseudogenes
What are pseudogenes?
Nonfunctional gene copies
Non-expressed copies of genes
Hemophilia - caused by?
Caused by L1 insertion into clotting factor VIII gene
Adenomatous Polyposis Coli - causes colon cancer via?
Insertion of L1 into APC region of cancerous cells; not seen in healthy cells
Positives for Repetitive DNA
1) Promotion of gene repair = use copy after ds Break
2) Gene duplication = misalignment at single repeat
3) Gene deletion = misalignment of repeats
Centromeres
Considered satellite DNA
Monomeric sequences = 50-70% homology
Confer chromosome specificity
What are minisatellites?
Considered variable number of tandem repeats (VNTR)
Shorter regions of repeating elements (1-5kb)
>9 nucleotide length
What are microsatellites?
- Considered variable number of tandem repeats (VNTR)
- Referred to as short tandem repeats (STR) or simple sequence repeats (SSR)
- 1-8 kb long
Central Dogma of Molecular Biology
DNA —> RNA —> proteins
Name and classification
Adenine
Purine
Name and classification
Guanine
Purine
Name and classification
Cytosine
Pyrimadine
Name and classification
Thymine
Pyrimidine in DNA
Name and classification
Uracil
Pyrimidine in RNA
Name
Symbol
Nucleoside
Adenylate (Adenosine-5’-monophosphate)
AMP
Adenosine
Name
Symbol
Nucleoside
Guanylate (Guanosine-5’-monophosphate)
GMP
Guanosine
Name
Symbol
Nucleoside
Uridylate (Uridine-5’-monophosphate)
UMP
Uridine
Name
Symbol
Nucleoside
Cytidylate (cytidine-5’-monophosphate)
CMP
Cytidine
Name
Symbol
Nucleoside
Deoxyadenylate (deoxyadenosine-5’-monophoaphate)
dA, dAMP
Deoxyadenosine
Name
Symbol
Nucleoside
Deoxyguanylate (Deoxyguanosine-5’-monophoaphate)
dG, dGMP
Deoxyguanosine
Name
Symbol
Nucleoside
Deoxythymidylate (deoxythymidine-5’-monophoaphate)
dT, dTMP
Deoxythymidine
Name
Symbol
Nucleoside
Deoxycytidylate (Deoxycytidine-5’-monophoaphate)
dC, dCMP
Deoxycytidine
Backbone of DNA formed by
Phosphodiester bond
Covalent bond
Which ends are involved in DNA backbone and what product is there
5’ phosphate group
3’ hydroxyl group
Bonding between DNA strands
Hydrogen bonding
Chargaff’s Rule
(A+T)+(G+C) = 100%
Structure of DNA
double stranded
Anti-parallel to one another
Strands will complement one another
bonds connecting A —> T
2 Hydrogen bonds
bonds connecting G —> C
3 hydrogen bonds
Which nucleotide bases are easiest to break apart?
A to T
Only double bonds present
What forms backbone of DNA
Sugars (ribose/Deoxyribose) + phosphates
What part of DNA faces out?
Hydrophilic phosphodeoxyribose backbones
What faces towards the interior of helix?
Nucleotide bases
How many bases per helical turn?
10.5
36 angstroms
Function of major/minor grooves in DNA
Provide binding sites for regulatory proteins
Importance of Cisplatin
Anti-cancer drug
Will intercollate between DNA strands
Prevents DNA polymerase from continuing with replication
Results in apoptotic death
Distance between nucleotides of backbone?
3.4 angstroms PER base
Various forms of DNA and where seen?
B-form: normal presentation; most stable
A-form: dehydrated B form; protection in bacteria
Z-form: L handed DNA; GC rich sequences; play role in gene regulation
DNA synthesized by?
DNA Polymerase
DNA synthesis - strand is read in which direction?
5’ to 3’
With addition of nucleotide to DNA backbone, what molecule is released?
Pyrophosphate
Pi-Pi
DNA polymerase requires what to begin synthesis of new DNA strand
DNA primer and template primers
Primer DNA provides?
Terminus 3’ -OH group
Template DNA primers provide?
Sequence specifying complement sequence for DNA chain
Importance of Azidothymidine Zidovudine?
Antiviral medication for HIV
Incorporates itself into viral DNA
Unable to continue replication due to no terminal -OH group on 3’ end
Why is DNA replication considered semiconservative?
One parent strand and one daughter strand
DNA is ALWAYS synthesized in what direction
5’ to 3’
Which strand of DNA will continuously form new DNA strand?
Leading strand
Which strand of DNA will NOT continuously form a new strand?
Lagging
Leading strand is always synthesized in what direction
Towards the replication fork
Lagging strand is always synthesized in what direction?
Away from the replication fork
Short pieces of newly replicated DNA on lagging strand
Okazaki fragments
What splices Okazaki fragments together on lagging strand?
DNA Ligase
How does DNA proofread itself during replication?
Constantly scanning termini of nascent DNA chains
How does DNA go about correcting errors in nascent DNA strands
3’-5’ exonuclease activity of DNA Polymerase
Which part of cell cycle checks whether cell is ready for replication?
G1 checkpoint
What are some factors that cells might assess for at various checkpoints?
Size
Nutrients
Molecular signals
DNA integrity?
Where are the normal replication checkpoints in cell cycle?
G1
G2
Metaphase
Function of Retinoblastoma protein
Binds to E2F transcription factor
Prevents transcription of proteins - prevents cell cycle progression
What releases pRb from E2f?
Being phosphorylated by a kinase
When E2F released from pRb, what happens?
E2F will bind to DNA and begin transcription of proteins
Begins progression of cell through cell cycle
What does E2F transcribe for?
Cyclin E and DNA Polymerase
DNA replication begins where?
Origin of replication
What recognizes Origin of Replication?
Origin of replication binding proteins
of origins of replication in eukaryotes
Multiple
of origins of replication in prokaryotes
One
Initiator proteins
ORC
Cdc6
Cdt1
What do initiator proteins do?
Recruit replicative helicase to bind to DNA
What forms replicative helicase in eukaryotes?
Cdc45
Mcm2-Mcm7
GINS complex
CDC6 and Cdk2 - Importance in terms of regulation during G1 and S phases
G1: Cdk2 activity low, CDC6 accumulates; pre-RC forms but not active
S: Cdk2 activity high, CDC6 inactive; pre-RC is activated
Why is regulation of CDC6 and Cdk2 important?
Ensures DNA replication happens only ONCE per cycle
ORC proteins function?
Recognize origin of replication site
Topoisomerase function?
Relieves supercoils from DNA ahead of replication fork
Mcm function?
DNA Helicase that unwinds parental duplex
Cdc6, Cdt1 function?
Unwinds DNA
Loads Replicative helicase onto DNA
RPA/SSB functions?
Maintains DNA in single stranded state (single stranded binding proteins)
RFC function?
Subunits of DNA holoenzyme; load clamp onto DNA
DNA Polymerase Delta/epsilon functions?
Primary replication enzymes; Synthesize entire leading strand and Okazaki fragments
Is able to proofread DNA strands
PCNA function?
Ring shaped subunit of DNA Polymerase holoenzyme
Clamps replicating polymerase to DNA
Works in conjunction with DNA Polymerase III (bacteria) and Pol Delta/Epsilon in Eukaryotes
Primase function?
Synthesize RNA primers
DNA Polymerase alpha function?
Synthesize short DNA oligonuclotides as PART OF RNA-DNA primer
DNA Ligase function?
Seals Okazaki fragments to one another; forms continuous strand of lagging strand
FEN-1/RNAase H functions?
Removes RNA primers
Possible to attach RNA primer to end of lagging strand?
No
Result of inability to attach RNA primers to end of lagging strand?
Lose a little DNA each time cell divides
Telomerase function?
Reverse transcriptase that add telomeric DNA to telomers
- RNA-dependent DNA polymerase
- Prevents shortening of DNA strands
Function of telomers?
Maintain genomic integrity
Prevent nucleases from “attacking” chromosomes
Successive shortening of telomers causes?
Chromosomal instability
Chromosomal instability can lead to?
Cell senescence or apoptosis
Why is telomerase important for cancer cells?
Maintains continuous telomere length; cell never realizes it needs to die because length is consistently maintained
Classifications of different studies in research?
Descriptive
Analytical
What is descriptive research?
Does not seek to quantify relationship; only means to give an overview of what is happening in a population
What is analytical research?
Seeks to quantify the relationship between 2 things
Seeks to understand effect of intervention/exposure on OUTCOME
Examples of Descriptive Studies?
Case Reports
Case Series
Qualitative Studies
Surveys (cross sectional)
Subtypes of Analytical Studies?
Observational
Experimental
Observational Studies
Researcher is passive with involvement
Experimental Studies
Researcher is more active with involvement
Describe a cross-sectional study?
- Refers to a specific point in time
- Snapshot at THAT moment in time
- Can determine Prevalence
(# persons affected/# persons studied)
Describe a case-controlled study?
- Compares group of participants possessing condition of interest with similar group lacking condition
- Used to study rare incidences or outcomes
Describe a cohort study?
- Observational study following a group of people over time
- Examines how certain factors affect health outcomes
- Used to determine incidence
(New cases/population)
Describe Prospective cohort study?
- Observational study focused on following group of people over time
- Will collect data on exposure to factor of interest
- Outcomes are tracked to see if association between exposure and outcome
- Looks FORWARD in time to see relationship between exposure and outcome
Describe Retrospective cohort study?
- Observational study focusing on individuals with exposure to disease/risk factor
- Analyze health outcomes over time to form connections/assess risk of outcome with given exposure
- Look BACK in time to examine relationship between exposure in past and present outcomes
Advantages/Disadvantages of Cross-sectional study?
Advantage: Quick, easy
Disadvantage: No cause and effect
Advantages/Disadvantages of Case-controlled study?
Advantage: Good for rare disease; able to generate hypothesis
Disadvantage: Establish correlation, not CAUSATION; Recall bias
Advantages/Disadvantages of Cohort study?
Advantage: Establish cause and effect
Disadvantage: LONG Follow Up; Loss to follow up; Expensive
Describe Experimental Study
Interventions used
Investigator controlled maneuvers
Types of Experimental Studies?
Randomized trials
Non-randomized trials
Quasi-experimental
What measure of association corresponds to Cohort studies?
Relative risk
What measure of association corresponds to Case-Control studies?
Odds ratio
What measure of association corresponds to Cross-sectional studies?
Odds ratio
Prevalence ratio
Odds ratio
Odd’s of disease in exposed/odds of disease in UNexposed
- Odds outcome occurs with exposure compared to odds of outcome occurring without exposure
Relative Risk
Risk of disease in exposed/risk of disease in UNexposed
When does odd ratio = relative risk?
If disease is rare
Which types of studies give the strongest evidence?
Meta-analyses and Systematic Reviews
Which types of studies give the weakest evidence?
Cohort Studies
Case controlled studies
Case report
Which types of studies give medium strength for evidence?
Randomized control studies
Quasi-experimental
Advantages of Medical Documentation?
1) Continuity of Care
2) Allows provider to provide thinking process/management plan clear to all
3) Longitudinal pic of overall health of person
80% of diagnoses made on _______ alone.
Patient history
Why is H&P important?
1) Convey concise/detailed info about patient hx and exam findings AT TIME
2) Outlines plan to address issues
3) Way of communication with providers
4) Medico-legal documentation
Part of Patient History?
CC
HPI
PMH
FM
SH
ROS
Parts of Patient Physical Assessment?
1) Current and thorough head-to-toe exam
2) Can also include mental status exam
3) KNOWN and relevant labs/imaging results
Parts of Assessment?
1) ID/localize abnormal findings
2) Interpretation of findings
3) Make a hypothesis about issue
4) Formulate Differential Dx
5) Generate problem list with CC and active issues at top
Parts of Plan?
Inclusion of evaluation and/or management of problems
SOAP notes
Subjective
Objective
Assessment
Plan
Use of the SOAP note
Organizing patient information
Daily updates
Focus on “active” problems
Focused history and PE
S in SOAP note:
Meaning
Info in section
Subjective
What patient tells you
Hx of symptoms/CC/HPI
RELEVANT PMH, FH, SH, and ROS
O in SOAP note:
Meaning
Info in section
Objective
What YOU observe (hear, smell, feel, see)
PE findings/mental status changes
Available XR/Lab results
A in SOAP note:
Meaning
Info in section
Assessment
What YOU think is going on
Problem list/differential dx
P in SOAP note:
Meaning
Info in section
Plan
What YOU are going to do
Work up and/or management plan
How long are medical records maintained
Depends on state law - usually no longer than 10 years.
Allowed to refuse to give patient records to patient
No - not for any reason
Items of info to share with patients
Medication List
Vital Signs
Trends related to chronic illness
Patient education material
What to include in patient notes?
Remain neutral and professional
Avoid hostile and derogatory statements
Allowed to change medical record?
No - allowed to make an addendum
Ways to avoid errors
Do not use drug abbreviations
Don’t use hanging zeros
Can use LEADING zeros
Issues with medical dictation?
Speech recognition STILL makes mistakes - go back and proofread notes before completing them
Issues with telemedicine?
Social distancing
Isolating for provider
Patients feel less connected to provider
Document phone calls?
Yes - need to be recorded for legal and medical reasons
Define: Exogenous sources of genetic damage
Influences from outside environment
(UV light, radiation, carcinogens)
Define: endogenous sources of genetic damage
Unintended consequences of metabolic processes
(Oxidation, nitrosylation, hydrolysis)
Why is UV light so potent and exogenous source of damage to DNA?
- Will form covalent bonds between pyrimidines bases
- Form pyrimidine dimers
- Distort DNA structure - unable to replicate correctly
Define: Substitution mutations
Change in ONE DNA base
CaDefine: Transitions
- Change in base pairs
- Purine replaced with another purine
- Pyrimidine replaced with another pyrimidine
Define: Transversions
- Change in base pairs
- Purine replaced with pyrimidine
- Pyrimidine replaced with purine
Define: Indels
Insertion/deletion of SINGLE NUCLEOTIDE PAIR!
Define: Silent mutations
- Change in codon that doesn’t affect final product
- Usually occurs in the Wobble site of codon
Define: Missense mutations
- Mutation resulting different AA inserted
- Can change whole property of protein
Define: Nonsense mutations
- Premature stop codon inserted in wrong position
Define: Frameshift mutations
- Due to 1-2 base deletions/additions
- Changes AA sequence
- Result in shortened proteins
Define: Splice Site mutations
- Formation/deletion of splice site for introns/exons
- Acceptor site (3’ end) = scan for next sequence; exon removed
- Donor site (5’ end) = scan for next sequence; introns remain
Define: Loss of function
Proteins that are quickly degraded or Nonfunctional proteins
Define: Gain of function mutations
Protein bind to something it should not
Define: Genomic instability
Increased propensity for genetic mutations
Why is Methyl-C mutation hot spot?
- Methylated Cytosine tells cells which is parent strand and which is newly synthesized strand
- Needed for repair mechanism to function properly
Germline mutations:
Parental gametes
1) Passed to offspring
2) Can result in disease
3) Genetically inherited diseases
4) Every cell of individual contains mutation
Germline mutations
Embryo
NA
Somatic mutations:
Parental gametes
N/A
Somatic mutations:
Embryo
Cannot be passed onto offspring
Somatic mutations:
Organism
1) Occur in single cell
2) Produce clones with mutation
3) Result in phenotypically mutated region of body
4) Frequently involve CA
Germline mutations:
Organism
Mutation present throughout organism
Germline mutations:
Offspring gametes
1) Result in inherited disease
2) Present in 1/2 gametes produced
3) Ex: NF-1 gene
Somatic mutations:
Offspring gametes
1) Limited to somatic cells
2) Not present in gametes
3) Not genetically inherited
4) Ex: McCune-Albright Syndrome
Father Effect concerning mutations
- Higher likeihood for mutation due to number of times chromosomes divide
- Begin around 15 yrs - chromosomes will divide around 2,000,000 times by 50 yo
Mother effect concerning mutations
Direct Repair of DNA
- Repair of DNA without breaking of phosphodiester bonds
- Addresses spontaneous methylation
- Ex: O6-methylguanine-methyltransferase
Single-strand Repair of DNA
- Lesion on one strand of DNA helix
- Will use complementary strand as template for repair
Base Excision Repair of DNA
- Lesions affecting single nucleotide
- Introduction of uracil in DNA chain, deamination (alter base) and depurination (lose base)
- Caused by endogenous sources
Nucleotide Excision Repair of DNA
- DNA lesion involving more then 1 nucleotide
- Effect of foreign molecule
- Removes/replaces larger sections of nucleotides to fix lesions
-Cleaved by endonucleases
Post-Replication Mismatch Repair of DNA
- Incorrect base inserted into DNA strand and DNA polymerase didn’t catch it
- Mut proteins recruit endonucleases to remove/replace section with mismatch nucleotide
Homologous Recombination
- Use homologous chromosomes as template to repair severed strands
- Occur during S and G2 phases of cell cycle
Nonhomologous End Joining
- Special ligase fuses ends of 2 DNA fragments
- Will connect any 2 ends of ANY 2 severed strands it finds
- HIGH ERROR prone
- Results in translocations
Types of RNA: mRNA
Messenger RNA
Proteins
Types of RNA: tRNA
Transfer RNA
Used in protein translation
Types of RNA: rRNA
Ribosomal RNA
Forms scaffold of Ribosome
Types of RNA: snRNA
Small nuclear RNA
Used in ribonucleoproteins (spliceosomes and telomerase)
Types of RNA: hnRNA
Heterogeneous nuclear RNA
pre-RNA - single strand of immature mRNA
Types of RNA: miRNA
MicroRNA (ssRNA)
Fragments of nascent RNA’s
Formed via spliceosomes
Inhibitors of translation
Types of RNA: siRNA
Silent Interfering RNA
Viral origins (dsRNA)
Cleaves mRNA
Will down regulated gene expression
Types of RNA Polymerase:
RNA Polymerase I
rRNA in nucleolus
Types of RNA Polymerase:
RNA Polymerase II
mRNA in nucleus
Types of RNA Polymerase:
RNA Polymerase III
tRNA and other RNA’s in nucleus
Types of epigenetic changes to DNA
Methylation of histones. cytosine
Acetylation/Phosphorylation of histones
With epigenetic changes, what happens to DNA
Methylation - condenses
Acetylation/Phosphorylation: loosening of nucleosomes
When nucleosomes relax, what can bind to DNA?
DNA binding proteins
DNA binding proteins are result of
End products of 2nd messenger systems
Example of DNA binding proteins
E2F
Protein bound to E2F to keep in inhibited?
Retinoblastoma protein
What causes pRB to release from E2F?
Phosphorylation via kinases
Location of enhancer region for DNA
Upstream of binding site - towards 5’ end
Location of repressor/silencing region
Just upstream of binding site
TATA box
Promotor region for binding
Location of primary transcript to be read?
Downstream of promotor region - towards 3’
Types of controls enhancers/silences have?
Tissue specific
Time specific
Organ specific
Cell type specific
Role of Transcription enhancers
Enhance transcription of gene set
Force nucleosome rearrangement to allow promotor region to be accessible
Role of Transcription silencers/repressors?
Prevent transcription
Mechanisms by which transcription repressors block transcription?
1) Obstruction of promotor site
2) Force nucleosomal rearrangement to prevent RNA Pol from binding
3) Bind protein that obstructs promotor either directly or indirectly
If E2F is bound to DP1 - what occurs?
Looping of DNA
Looping of DNA allows for what?
Allow enhancer element further down to interact with transcription-initiator complex
What proteins are regulated by E2f being bound to DP1?
CDC6
MCM
DNA Pol delta
All seen in late G1
What is DP1?
What does it form with E2f?
Stimulates?
Represses?
1) TFDP1
2) Will form heterodimer with E2f
3) Stimulated E2F-dependent transcription
3) Represses cell-cycle dependent genes in quiescent cells
4) Inevitably activates G2/M genes
What is TFIID?
Complex of TATA Binding protein abd TATA box protein associate factors
Direction DNA read?
Read from coding strand in 5’ to 3’ direction
DNA equivalent to RNA produced?
Coding strand (exchange T for U)
All else remains the same
Template Strand
Bound by?
Used for?
Bound by RNA Polymerase
Used for base pairing nucleotides during transcription
RNA polymerase requires 3 things:
1) Nucleosome moved out of way
2) Transcription regulators to bind to DNA
3) Transcription factors bind regulators and DNA AT start site
All transcripts will have 3 regions?
5’ UTR
3’ UTR
Coding region
TATA Box:
Sequence found in core promotor region
Located 25-35 bp upstream from start site
CCAAT box
- GGCCAATCT
- Consensus sequence upstream 60-100 bp to initial transcription site
- Signals binding site for RNA transcription factors
Pribnow box
Bacterial equivalent of TATA box
Shorter sequence
Proteins that initially binds to RNA Polymerase II promotors?
TBP and TFIIA
Subsequent proteins that bind to RNA Polymerase II binding site
TFIIB w/ Polymerase II
TFIIE
TFIIH
RNA Polymerase II Binding proteins form what type of complex with one another
Closed - not activated yet
In the RNA Polymerase II Binding protein complex, where is DNA unwound?
The Inr region
What enzyme/activity unwinds DNA at the INR region of the RNA Polymerase II Binding protein complex?
Helicase
Which RNA Polymerase II Binding protein has the activity of helicase?
TFIIH/THIIE
(Allows RNA Pol II to begin reading DNA)
Once DNA unwound at Inr region, what does this form?
Open complex
What end of Pol II protein is phosphorylated by THIIH?
Carboxyl-terminal end (CTD)
What does phosphorylating Pol II protein do for transcription?
Allows polymerase to escape promotor and begin transcription
What comprises the Carboxy Terminal Domain of RNA Pol II?
52 repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser
What is the Carboxy Terminal Domain involved in?
Initiation of transcription
5’ Capping of transcript
Attachment of spliceosome for splicing
Which residue in Carboxy Terminal Domain get phosphorylated to being capping of transcript and bring factors to form Poly A tail?
Ser5
What phosphorylated Ser5 in the Carboxy Terminal Domain?
THIIH
What Serine is phosphorylated to actually ACTIVATE elongation of transcript?
Ser2
Termination of elongation occurs via?
Dephosphorylation of RNA Pol II
Initial activation of RNA Polymerase II occurs where?
Final activation of RNA Polymerase II occurs where?
Serine 5 residue
Serine 2 residue
RNA Pol II will only work with?
RNA nucleotides
RNA Nucleotides
ATP, GTP, CTP, UTP
First nucleotide of RNA transcript placed where in relation to promotor/TATA box?
Just 3’
After first few nucleotides placed, what replaces initial transcription factors?
Other transcription factors that promote elongation
What is the D-loop structure?
Where 2 stands of dsDNA molecule are seperated for a stretch and are held apart by a third strand.
Initiation stage of DNA replication completes when?
RNA is greater that 10 bp long
Hallmark sign of end of initiation
Capping enzyme binding to 5’ end and capping 5’ end
Introns
- Sequences of RNA removed to make mature RNA
- Non-coding regions of RNA
Exons
- Sequences of RNA retained in mature mRNA
- Coding regions of RNA
Structure that removed introns from immature RNA?
Spliceosomes
Spliceosome
- Large ribonucleoprotein complex
- Seen in nucleus of eukaryotic cells
Modifications of RNA occur when?
As transcript is made
Modification of tRNA and rRNA
Fold into mature 3D form as soon as RNA released from D loop
Polycistronic mRNA
mRNA that encodes for multiple proteins from a SINGLE gene
Modifications of mRNA
5’ capping - usually GTP
3’ Poly A tail
Removal of introns via spliceosomes
Modifications made to tRNA
Folded
Spliced via spliceosomes
3’ CCA ending added
Modification of rRNA
Folded
Spliced via spliceosomes
Formation of 5’ Cap on mRNA
- 7-methyl-guanosine
- Connected to RNA via 5’-to-5’ phosphate bridge
Functions of 5’ Capping?
Protect 5’ end of RNA
ID RNA as mRNA
Splicing of mRNA
- Intron sequence beginning with 5’-GU and 3’-AG sequence with A-branch point in sequence between
Donor splice site
5’ end of mRNA
Acceptor site
3’ end of mRNA
When does splicing occur?
At same time as transcription or immediately thereafter
What molecule attaches to 5’ donor site and 3’ acceptor site?
snRNP (spliceosome)
Intro binding proteins act in what way for splicing?
Bringing 3’ end of one exon close to 5’ end of another further down mRNA
How does splicing occur?
- 5’ end of intron excised - attached to Adenosine near 3’ end of intron
- Loop is formed at GpA area
- 3’ end of intron excised while exons ligated together
Functions of snRNP’s
1) Recognize splice site
2) Catalyze lariat site
3) Fuse exons together
Estimation of how many diseases are correlated to splicing errors?
1/3 of all disease
Calcitonin alternative processing
- Calcitonin gene related proteins formed in thyroid; not formed in brain
Regulation of splicing
RNA binding proteins and snRNA/spliceosomes
Alternative splicing forms?
Different isoforms of RNA
Exon skipping occurs due to ?
- 5’ end of intron spliced downstream of normal intron
- Removes exon between them
Intron retention occurs due to?
- Splice sites of intron repressed/lost due to mutation
- Retain intron in mature RNA
Truncated proteins formed by?
Change in reading frame of RNA
Alternate splicing results in?
Alternate protein sequence/function
Physiological importance of alternate splicing
BRCA1 gene in response to DNA damage
Point mutations can lead to?
Abnormal splice site
RNA structure
- Can form double helices within ITSELF
- A form
RNases
Enzymes with specificity acting on RNA
Maturation of RNA requires?
RNases
tRNA control of RNA activity:
3’ end
5’ end
- 3’ end modified by adding CCA; recognizable for aminoacyl-tRNA synthase
- 3’ end protected by addition of AA to 3’ -OH group
- 5’ end protected by base pairing
mRNA control of RNA activity:
3’ end
5’ end
- 5’ end capped with 7-methyl-guanosine
- 3’ end with Poly A tail
- Poly A Binding Proteins (PABP) protect 3’ end
- PABP required for transport OUT of nucleus
Function of Aminoacyl-tRNA synthetase?
Connects correct AA to tRNA via binding pocket hydrogen bonding at 3’ end of tRNA
(Uses ATP to charge AA to tRNA at 3’ end)
5’ end of tRNA trimmed by ______ for what purpose?
RNases
Has single 5’ phosphate left
3’ end of tRNA trimmed by ______ for what purpose?
- tRNAse Z
- Non-coded CCA added via nucleotidyl-transferase
Middle loop of tRNA
Anti-codon loop
Where complementary sequence is encoded
Start codon
AUG
Stop Codons
UAA
UAG
UGA
How many codons encode for AA’s
64
How many AA’s are produced?
20
Redundant nature of genome
- Multiple codons coding for ONE AA
- Allows for wiggle room at Wobble site (3rd codon)
Point mutation causes?
Deamination or methylation of DNA bases
Which DNA strand is match for RNA strand when translated?
Coding strand of DNA
What nucleotide is replaced in RNA from DNA
Thymine become Uracil in RNA
Coding sequence
Replace every T with a U
Everything else remains the same
Reading template sequence?
Form reverse compliment in RNA form
5’ end is on right!
Translation of mRNA
- mRNA MUST be matured
- Must exit nucleus
- Ribosome properly loaded to find AUG codon
- Elongation using charged tRNA’s bound to E2F
- Termination at stop codon
How is mRNA removed from nucleus?
Nuclear Export Factors and TF’s
PABP functions?
1) bind 3’ poly a tail
2) bind TF’s to 5’ cap
3) Cause mRNA to circularize to exit nucleus
Pre-initiation of translation beings where?
Nucleus
Initiation factor that binds 5’ cap of mRNA?
eIF4F
eIF4F role?
Promote eukaryotic translation initiation
Composition of eIF4F?
- Heterotrimeric protein complex
- Consists of eIF4A, eIF4G, eIF4E
Function of eIF4A?
Helicase activity
Function of eIF4G?
Scaffold protein between eIF4F complex and PABP
Function of eIF4E?
Cap-binding protein
Which ribosomal subunit will eIF4F bind to?
40S, smaller subunit
Key protein in control of polypeptide chain initiation?
Guanine nucleotide exchange factor (eIF-2B)
Role of eIF-2B?
- Exchanger of GTP for GDP
- Forms ternary structure (eIF-2.GTP.Met.tRNAf)
Function of eIF-2.GTP.Met.tRNAf structure?
Initiator complex for translation
If stressors act on eIF2B complex?
- Phosphorylated alpha subunit of complex
- Will inactivate complex
If eIF2B inactive, what happens to protein synthesis?
Decreases
IRES region
Internal Ribosome Entry Site
Pre-initiation looping of?
Poly A tail - PABP - eIF4G - eIF4A - eIF4E
If eIF4G lost/phosphorylated, what occurs?
Initiation of translation is aborted
Ways to lose eIF4G
1) Cleavage by Capsase 3 during apoptosis
2) Cleavage by viral proteases
Alternate path for eIF4G to be activated?
Using Internal Ribosome Entry Site (IRES)
Activation of IRES site?
Proteolyzed or modified eIF4G
Virus that can use IRES site to advantage?
Picornaviruses
Mechanism of action by picornaviruses on eIF4G?
Proteolyze eIF4G
Virus has IRES that will override host mRNA
Steps for elongation:
Step 1
Step 1: Met-tRNA at P site
Steps for elongation:
Step 2
2nd tRNA binds at A site on 60S ribosomes
Guided by eEF1
Steps for elongation:
Step 3
Peptide bond formed between Met and other AA
Steps for elongation:
Step 4
mRNA and tRNA complex shifted to R (frees up A site)
Steps for elongation:
Step 5
- tRNA in E site ejected
- Peptide bonds form between Chain and new AA in A site
Steps for elongation:
Step 6
mRNA and tRNA complex shift again
Free up A site
Function of eEF2 during elongation?
- Catalyze hydrolysis of GTP
- Provides energy for translocations from A to P sites
Termination factors bind to ribosome at what time?
Encountering a stop codon
(UAA, UGA, UAG)
Enzyme that attaches AA’s on peptide chain
Peptidyl- transferase
Composition of peptidyl-transferase
Composed entirely of RNA
Ribozymes
Enzymes not made of proteins but ribonucleotides
Types of epigenetic regulation?
1) Methylation
2) Acetylation
3) Phosphorylation
4) Change in nucleosomal structure
Changes caused by methylation
- Addition of methyl group to DNA
- Represses transcription
Changes caused by acetylation?
- Occurs at histones
- Allows for increased availability of DNA
- Increased transctiption
Changes caused by phosphorylation?
- Occurs at histones
- Allows for increased availability of DNA
- Increased transctiption
Changes caused by nucleosomal structure changes
- Modulate availability of DNA to TF’s
Other means to control gene expression?
1) Temporal controls
2) Spatial controls
3) Environmental controls
4) Change in [metabolites] or [energy]
Decreased levels of ATP or GTP can lead to what for replication?
1) Cause delays/prevent cellular processes
1.1) Prevent replication due to not enough energy
1.2) Reduce transcription/translation - delay process, don’t load components
Role of ATP/GTP in replication/transcription/translation?
Energy transfer and biosynthesis rxns
Main event of G1 checkpoint?
Phosphorylation of Cdc6
Why is phosphorylation of Cdc6 important for replication?
It is the start signal of replication initiation
Consequences of low ATP levels in replication?
tRNA not charges
RNA polymerase hesitates when incorporation Adenine
Consequences of low GTP level in replication?
1) eIF2 unable to load 60S ribosome
2) eEF1 and eEF2 unable to load new aminoacyl-tRNA
3) eEF1 and eEF2 unable to translocate ribosomes
Enzymes which use ATP and GTP:
1) Histone acetylase
2) Topoisomerase
3) Helicases
4) FEN1
5) Ligases
6) Capping enzymes for mRNA
7) Spliceosomes
Enzymes which use ATP and GTP:
1) Nucleotidyltransferase
2) Aminoacyl-tRNA synthase
3) Translation initiation factors
4) eEF1 placing charged tRNA in A site
5) eEF2 in translation translocation
6) Translation Release factor
Function of FEN1
Maintain stable telomers
Function of nucleotidyl-transferase?
Part of repair pathway for single nucleotide base excision
During protein elongation, what is used as primary energy source?
GTP and ATP
Which parts of protein synthesis are GTP and ATP used for?
Translation initiation
Elongation
Termination
Tight control of protein synthesis highlights?
Importance of tight control over accurate processes and their energy usage (no energy wasted)
Crucial step in DNA replication
Assembly/activation of pre-Replication Complex (pre-RC)
What assembles pre-Replication complex?
CDK and DDK phosphorylation
During replication initiation: Cdc6 is phosphorylated by
CDK2
What happens to Cdc6 when phosphorylated?
Is removed from pre-RC
What takes places of Cdc6 in pre-RC?
Cdc45
With addition of Cdc45, the pre-RC becomes?
Initiation complex
PCNA
Proliferating cell nuclear antigen
What is responsible for loading PCNA on DNA?
Replication Factor C
Role of PCNA?
Sliding clamp holding polymerase on template strand
What is DDK?
Ser-Thr kinase
Composed of Cdc7 and Dbf4 subunits
What phase of cell cycle is DDK most abundant at?
S phase
ORC2L and ORC6L
Origin recognition complex subunits
Recognize origin of replication
Why does Cdc7 need to interact with Dbf4?
ATP binding
Substrate recognition
Ways transcription is regulated
1) Availability of nucleotides
2) Availability of binding sites for TF’s to bind to
Circadian regulation involves?
Glucocorticoid receptors
Heat shock proteins 70 and 90
Protein FKBP4
Hormonal regulation
Direct binding of hormone
2nd messenger signal in cascade
Vitamin D:
Binds to?
Mode of action to DNA?
Forms what?
Forms complex that binds to DNA
Rearranges nucleosomes, allowing RNA Pol II to bind
Transcribes Calbindin gene
Increased Calbindin expressions lead to?
Increased Ca2+ uptake in intestines
Adrenaline:
Binds to?
Mode of action to DNA?
Forms what?
Binds to GPCR
Triggers cascade effect - increased production of glycolytic enzymes
Allows cell to rapidly produce energy
Transcript Maturation:
RNA Pol II and its role
C-terminus is phosphorylated
What is RNA interference?
Post-translational gene silencing
How is RNA interference mediated?
Small RNA molecules called microRNA’s (miRNA)
How do miRNA’s function to silence?
Bind to complementary sequence on mRNAs
Either degrades mRNA or inhibits translation
RNA binding protein that increases RNA stability
HuR
Increased RNA stability leads to
Increased translation
RNA binding protein that increases RNA stability as well as transports RNA out of nucleus?
PABP
RNA binding protein that decreases RNA stability?
RPB AUR1
Mechanism of action for RBP AUR1
Binds to AU-rich sequences in 3’ UTR
Composition of miRNA
Smaller components of larger heteronuclear RNA
Pharmaceutical inhibitors of DNA replication
Acyclovir
Pharmaceutical inhibitors of Polymerases:
1) Cytarabine
2) Floxuridine
3) Fludarabine
4) Gemcitabine
Pharmaceutical inhibitors of dTTP production:
1) 5-Fluorouracil
2) 6-mercaptopurine
3) Capecitabine
4) Floxuridine
5) Fludarabine
6) Hydroxycarbamide
7) Methotrexate
8) Pemetrexed
Antibiotics that inhibit 30S small subunit
Aminoglycosides
Will bind to 30S subunit; defective product produced
Antibiotics that inhibit tRNA:
Tetracyclines bind to 30S unit
Inhibit bond formation of tRNA’s = no protein synthesized
Antibiotics that inhibit 50S large subunit:
Chloramphenicol, Lincoamides, Macroglides
Bind to 50S unit
Inhibit peptide bond formation = no protein synthesized
Method of action for Polio virus in transcription/translation?
- Will proteolyze eIF4G = prevents reinitiation of host mRNA containing 5’ caps
- Gives viral RNA preferential treatment for translation
Method of action for ricin toxin in transcription/translation?
Breaks glycosidic bonds in ribosomal subunits
Results in no protein synthesis because adenine removed from 28S rRNA
(Overall peptidyl-transferase inactive)
Method of action for Diphtheria toxin in transcription/translation?
From Corynebacterium diphtheria
A subunit of toxin processed by endosome and release in cytosol
- Toxin will ADP-ribosylate E2F
- E2F unable to catalyze GTP dependent translocations
- Translation ceases because A site cannot move to P site and P site cannot move to E site in ribosomes
Define: alleles
different versions of a gene
Define: Gene
1) Sometimes can be transcription unit
2) Sometimes can be allele of chromosome
Define: Genotype
Genetic composition of a person
Define: Phenotype
Physical presentation of a persons genotype
Define: Homozygous
Two alleles are identical
Define: Heterzygous
Individual with 2 different alleles
Define: Hemizygous
Only ONE copy of an allele
Define: Haploinsufficiency
Only one copy of allele delivers insufficient amount of product
Define: Gamete
Either egg or sperm; sex cells
Define: Affected
Person WITH symptoms of disease
Define: Congenital
Symptoms present at birth
Define: Fitness
Number of offspring reaching reproductive age / average number for population
Define: Penetrance
Proportion of individuals with mutation exhibiting clinical symptoms among all individuals with such mutation
Define: Expressivity
Degree to which phenotype expressed by individuals with particular genotype
Define: Variable Expressivity
Difference in severity or age of onset for seemingly identical genotype
Seen mostly in dominant diseases
Define: Pleiotrophy
One gene influences 2+ unrelated phenotypic traits
One gene = mutliple phenotype results
Define: Allelic Hetergenicity
Different mutations at same location on allele lead to same/similar phenotypes
Define: Locus heterogenicity
Mutations at MULTIPLE gene locations capable of producing SAME phenotype
Each mutation sufficient to cause phenotype
Define: Phenocopy
Variation in phenotype caused by environmental conditions
Organisms phenotype matches phenotype determined by genetic factors
*not a mutation
Define: Genetic marker
Gene or DNA sequence with known location on chromosome and can be used to ID individual/species
Define: Delayed age of onset
Presentation of disease delayed until later in life
Which inheritance pattern is more common than most?
Heterozygous
Which inheritance pattern is less common than most?
Homozygous
Most common inheritance pattern in marriages
Heterozygous marries homozygous
Fitness in dominant diseases:
reduced fitness –> expected lowered incidence in next gen
Autosomal Recessive
1) Only homozygous carriers demonstrate symptoms
2) Parents are usually heterzygous usually
Loss of function in what is typical in recessive inheritance?
Enzyme function
Compensation/1/2 amount of product is good enough
Loss of function in what is typical in dominant inheritance?
Structural proteins
1/2 amount of product NOT enough
Leads to haploinsufficiency
Gain of function occurs in which inheritance pattern?
Dominant inheritance
In a pedigree - what does proband refer to?
First family member who brought family to attention of person taking family history
What is indicated by red circle?
Person is desceased
What is indicated by red square?
Monozygotic twins
What is indicated by red circle?
Proband individual
What is indicated by red circle?
Dizygotic twins
Define: Autosomal dominant inheritance
Phenotype shows presence of allele
Also in Heterozygotes
Gene is on Autosome
Define: Autosomal recessive inheritance
Phenotype ONLY present when two disease causing alleles present
Recognizing AR:
1) Do not normally have affected parent
2) 25% of time affected in siblings
3) Parents often distantly related in some way
Recognizing AD:
1) All affected have at LEAST one affected parent
2) Average is 50/50 for siblings
Autosomal inheritance
Equal number males inherit as females
X-linked recessive inheritance
Phenotype is no other allele present
Hemizygous in males
Rarer in females
X-linked dominant inheritance
Always will show phenotype
Y-linked inheritance
Affects only males
Male fertility
Recognizing XR:
More males will be affected than females
Daughters will be CARRIERS only
NEVER WILL SEE Father to Son transmission
Affect uncle-nephews
Recognizing XD:
More females than males affected
1/2 sons affected & 1/2 daughters affected
NEVER WILL SEE Father to Son transmission
Recognizing Y-linked:
ONLY WILL EVER BE FATHER TO SON
If mother is heterozygous:
XR?
XD?
XR: 1/2 sons affected; 1/2 daughters carriers
XD: 1/2 sons affected; 1/2 daughters affected as well
If father is affected:
XR?
XD?
XR: all daughters carriers; sons not affected
XD: ALL daughters affected; sons are carriers
Define: sex-limited inheritance
Genes on autosomes
Dominant OR recessive
Influence traits expressed only in ONE of the sexes
*Diseases linked to either male or females - only expressed in ONE sex
(Uterine cancers in females, prostate cancers in males)
Non-penetrance of disease
Predisposing genotype but does NOT show symptoms
Someone who has the disease but does NOT show it
Mitochondrial Inheritance
From Female Carrier to ALL offspring
Allelic Heterogeneity:
Gene
Variation
Phenotype
1) Only ONE location causes issue; different alleles at ONE location
2) Two of more alleles
3) Same or different
Ex: CFTR for CF
Locus Heterogeneity:
Gene
Variation
Phenotype
1) Two or more genes cause issue
2) At least ONE bad allele per gene
3) Same
Ex: Xeroderma Pigmentosum/Congenital Deafness
Define: Codominance
Example of:
Refers to both allelic copies being expressed at the same time
Neither is recessive or masks the other
Ex: AB blood types
Define: Incomplete penetrance
Example of:
Someone has diseased allele but does NOT express the trait of that diseased allele - have a higher likelihood to DEVELOP that trait
Ex: BRC1 and BRCA2
Define: Pleiotrophy
Example of:
One gene affecting MULTIPLE phenotypic traits
Ex: PKU - multiple symptoms OF disease
Define: Genetic Anticipation
Example of:
Process by which there is INCREASED severity and DECREASED age of onset of disease in subsequent generations
Ex: Huntington’s Disease (will get disease at younger and younger ages with worsening severity)
Define: Locus heterogeneity
Example of:
Process by which DIFFERENT LOCI/mutations cause the SAME PHENOTYPE
(In theory - different disease processes cause similar presentation of ONE of the disease processes)
Ex: Marfanoid habitus can be caused be Homocystinuria, Marfan Syndrom, or MEN 2B
Define: Heteroplasmy
Example of:
One or more types of mitochondrial DNA accounting for variable expression of Mitochondrial disease
DNA is both normal AND MUTATED
Ex: Mycoclonic Epilepsy with Ragged Red Fibers (MERFF)
Define: Variable Expression
Example of:
AKA: expressivity
Situation where people have same disease genotype but DIFFERENT symptoms
Ex: 1) Marfan’s Syndrome
Ex: 2) Neurofibromatosis Type 1
Normal chest
No structural deformities or visible retractions
Barrel Chest
Increased AP diameter
Pigeon chest
Anterior displacement of sternum
Funnel Chest
Depressed lower sternum
Throacic-Kypho-Scoliosis
Raised shoulder/scapula, thoracic convexity, and flared interspaces
Three groups of respiratory muscles
1) Diaphragm
2) Rib cage muscles
3) Abdominal muscles
Muscles of inspiration
1) SCM
2) Scalenes
3) Pectoralis minor
4) Pectoralis major
5) Serratus anterior
6) External intercostals
Muscles of Expiration
1) Internal intercostals
2) External Obliques
3) Internal Obliques
4) Transversus abdominis
Conducting Portion of Respiratory System:
Nasal cavities
Pharynx
Larynx
Trachea
Bronchi
Bronchioles
Terminal Bronchioles
Respiratory Portion of Respiratory System:
Respiratory Bronchioles
Alveolar Ducts
Alveolar Sacs
Alveoli
Anatomy of R Lung
2 Fissures - oblique and horizontal
3 lobes: superior, inferior, and middle
Anatomy of L Lung
1 fissure - Oblique
2 lobes - Superior and inferior
Cardiac notch - where the heart resides
Lingula - most anterior portion of Superior lobe; right near apex of heart
