Cell Bio Quiz 9

What is alkylating?

the additional of alkyl chains

What is a vesicant? erythema?

A blistering agent, inflammation of mucous membranes

What is septic shock?

Sepsis is when there is an infection in a specific tissue site, that has now gone systemic and entered the blood stream, shock meaning that there is a dramatic drop in blood pressure and body temperature (not enough blood flow goes through the organs, and organ failure occurs)

Explain basic structure of DNA…

• Base pairs include A-T (2 H-bonds) and C-G(3 H-bonds), and are bound together by Hydrogen bonds, the different numbers of bonds help make bases specific for one another (do our DNA is complementary)

• The 2 chains are antiparallel, if we are at one strand on the 5’ end, on the other strand we’d be at the 3’ end

What does DNA polymerase need help with?

1. DNA needs to be melted or separated in order for DNA polymerase to do its job, this is something it can’t do, so another molecule needs to expose the bases.

2. It cannot initiate a brand-new strand of DNA, it can only elongate an existing strand (this is why we need a primer)

3. Can only create new DNA in the 5’ to 3’ direction, meaning if its creating a strand, its READING the template DNA from the 3’ to 5’ end because they’re antiparallel

What are origins of replication? What are consensus sequences?

• Origins of Replication: sites at which DNA replication occurs

  • Prokaryotes: with single circular DNA, there is only 1 very well-known origin

  • Eukaryotes: much longer genome, multiple origins that are less well known what their sequences are

• Consensus sequence: short DNA segments that are well conserved, and recognized by replication proteins, these are rich in A-T bases because fewer H-bonds keeping them together allows for less energy needed to start replication, these also exist across species of prokaryotes, in eukaryotes its less known

What are the replication origins of prokaryotic and eukaryotic DNA?

Prokaryotic: identified consensus sequence on the origin, where replication begins, it is separated, then DNA polymerase produces a daughter strand based on the template, leaving us with two interlocking loops of DNA

Eukaryotic: multiple sites where replication is initiated, not consensus sequences, replication bubbles occur (each with replication forks at the ends) and fuse together over time, leaving us with 2 copies of the DNA

What is an initiator protein?

they bind to the origin of replication and COULD be able to separate strands

Describe the differences between prokaryotic and eukaryotic initiator proteins…

Prokaryotic: DnaA - can separate strands

Eukaryotic: Origin replication complex (ORC) - does not separate strands (so we need another molecule)

What do Helicases typically do?

Melt or separate duplex DNA

• this requires energy (ATP)

• Prokaryotic = DnaB (unwinds 5’ to 3’)

• Eukaryotic = Mcm2-7 (unwinds 3’ to 5’)

leave us with separate single stranded DNA

What do SSBs typically do?

H-binds have a intrinsic property to re-anneal together, Single Stranded Binding proteins just bind and keep this from happening

What do gyrases typically do?

eliminate supercoiling that accompanies unwinding (2 kinds)

Another name for gyrases is topoisomerases, explain the 2 types of

Type 1 topoisomerase: creates a cut in ONE strand of the DNA, and then it is moved around to alleviate kinking, also there is no net energy expenditure

Type 2 topoisomerase: typically, targets points where the 2 DNA strands are crossing, cuts both strands of DNA, passed through the other part of the DNA then rejoined to alleviate supercoiling, this uses energy

• DNA gyrase is a type 2

How do interlocking loops of circular strands of chromosomes get un-locked?

type II topoisomerase

• long linear chromosomes are simple tangled, not interlocked

What exactly does DNA polymerase do?

reading the template in the 3’ to 5’ direction, adding nucleotides to the growing strand that is 5’ to 3’

What does it need to do this?

• template

• Deoxyribonucleoside triphosphates (DNTPs): source of nucleotides, as well as the energy source (phosphates on it)

• a primer

What is the primer?

a short sequence of nucleotides provided by primase (DNA-dependent RNA polymerase)

• short sequence of RNA is added (do not get this confused with an RNA primer, those can initiate new strands)

What is DNA polymerase III? (polymerase delta in eukaryotes)

synthesizes 5’ to 3’, but also proofreads 3’ to 5’

• excising incorrect nucleotides as it polymerizes (they cause physical disturbance in the strand)

• exonuclease: cleaves polynucleotide ends

• endonuclease: cleaves polynucleotides in the middle

Synthesizes the reverse complement of the parent

Processivity clamp function of DNA pol III or delta…

once it is bound, it clamps on there and doesn’t let go

Leading and lagging strands…

after the DNA is separated, the leading strand of DNA is replicating the direction of the replication fork where DNA will continue to separate producing a continuous strand of DNA, on the other strand once there is sufficient space available, replication starts there as well and produces choppy segments each requiring a new polymerase (Okazaki fragments)

• these are specific to replication fork

What is DNA polymerase 1 (or alpha in eukaryotes)?

Does the same things DNA pol III does…

• 5’ to 3’ polymerase

• 3’ to 5’ exonuclease

Also, uniquely exonuclease in 5’ to 3’ direction

• we cannot leave the RNA primer in the DNA, its temporary, DNA pol 1 removes the primer and replaces it with new DNA

slower than DNA pol III

Because of Okazaki fragments, and the primer being removed, we have segments of DNA that need to be joined together, how does this happen?

DNA ligase: repairs missing phosphodiester bonds between fragments

What are the differences in the entire system between prokaryotes and eukaryotes…

• gyrase vs. topoisomerase II

• DNA polymerase III vs delta

• DNA polymerase I vs. alpha

What is the end replication problem?

Linear chromosomes shorten by at least one primer length on each replicative cycle

• this is due to the fact that when a primer is removed, when there isn’t DNA on the other end for DNA polymerase to elongate off of at the ends, that primer doesn’t get replaced by DNA, therefore that DNA is lost on the replicated strand

What are the ends of our linear chromosomes called?

telomeres, hence, the end-replication problem gives rise to telomere shortening

What biological mechanism helps us to avoid telomere shortening?

Telomerase prevents the shortening of legging strands

• Telomerase is a RNA-dependent DNA polymerase with a build in RNA template

• main function is preventing shortening

Explain telomere erosion and cell senescence…

Telomere erosion leads to cell senescence: which is cell cycle arrest, where cells do not proliferate or grow, and subsequently no more DNA replication, a sign of aging

Pathogenesis of anthrax…

Macrophages phagocytose spores in the lungs, transport the bacterium across the epithelium, bacterium proliferates intracellularly, until cell lysis, then septic shock

What are antibiotics and antimicrobials?

Antibiotics: naturally made by some microorganism, against other microorganism, that we utilize to fight that microorganism as well

Antimicrobials: broader term, including both natural and synthetic substances that fight microorganisms

What are the desirable properties of an antibody?

• selectively toxic: microbe, not host (target features of pathogens that are unique to them)

• stable, cheap, easy to administer

• bactericidal (kill the bacterial) not bacteriostatic (not just stopping the growth)

• microbe shouldn’t be easily resistant

  • appropriate spectrum of activity

Explain spectrum of activity…

Broad spectrum can result in superinfection

• superinfection: a new infection complicating the course of antimicrobial therapy of an existing infectious process, resulting from invasion by bacteria or fungi resistant to the drug in use

start with narrow spectrum

Cipro (Ciprofloxacin)

• one of the most powerful antimicrobials available

• only effective treatment for inhalation anthrax

• it inhibits bacterial DNA gyrase (which doesn’t affect us)

Effects of mustard gas on replication…

• adds alkyl chains to guanines in your DNA

  • intra-strand crosslinks (stops DNA polymerase from continuing) |_|

  • inter-strand crosslinks (inhibits helicase melting of the DNA) between the two strands of DNA

• this leads to double stranded breaks in our DNA

Naturally occurring base damage…

• Guanine loses a purine (depurination event)

• cytosine becoming uracil (deamination event) RNA in our DNA!!!

• UV radiation can cause thymine dimers, linking adjacent thymine’s that inhibit replication

Base excision repair… (for damage on one strand of DNA)

• repair endonucleases recognize errors, usually detected by distortions in the helix

• damaged region is removed sometimes with the help of helicase

• polymerase fills the gap

• ligase repairs the backbone

Repairing double stranded breaks in our DNA…

• non-homologous end joining random sampling of nucleotides trying to repair it, will not be exactly the way it was before so it can lead to mutations

• homologous recombination: leverages the fact that there is another copy of the gene on the other chromosome, and uses that as a template to repair the broken strand, and leads to a perfect match

Cas9 and the CRISPR system for targeted double strand breaks

repeats in bacteria help protect them from viruses

• short palindrome loaded into a guide RNA, into Cas9 (an endonuclease)

• double stranded break is targeted, and the guide RNA is used as a template to rewrite the DNA

Diabetes mellitus is…

a group of metabolic diseases characterized by hyperglycemia(excess sugar in the blood) resulting from defects in insulin secretion, insulin action, or both

• insulin: when you consume food with sugar in it, the sugar is taken up across the epithelium in the small intestine into the blood stream, then that is now accessible to cells throughout the body, there are glucose transporters (ex: glut 4), insulin is secreted by beta cells, and binds to insulin receptors, that increased increases glucose transporter expression

other symptoms of diabetes

• polyurea: lots of uring

• polydipsia: constant thirst

• ketoacidosis: lipids become an alternative energy source for cells, ketone bodies are a byproduct of that, but they are acididc, and they build up causing dangerously high levels of acidity states in the blood or tissue

What are the 3 types of diabetes?

Type I: and autoimmune destruction of pancreatic B cells that secrete insulin

Type II: results from improper regulation of insulin or improper signaling

Type III: gestational, developed during pregnancy, will usually go away but if they’ve had it multiple times with different pregnancies, they can develop it permanently

Stages of Transcription:

1 transcriptional unit = 1 gene in prokaryotoes, but multiple genes in eukaryotes

• promotor and terminator sequences within the DNA itself

• RNA polymerase binds to the promoter, unwinding the DNA, reading it in the 3’ to 5’ direction, synthesizing an mRNA in the 5’ to 3’ direction

  • mRNA matches the coding strand almost exactly except there is uracil in the RNA instead of thymine

• RNA polymerase keeps moving along the template strand, elongating the mRNA, DNA behind it winds back together, until it reaches the terminator sequence

Promotors…

• the point at which the first nucleotide is bound is the start point, upstream from that is a consensus sequence containing promotor, that provides a signal for where RNA polymerase binds, conserved across species

• ex: TATA box is a critical consensus sequence within the promotor

Initiation…

RNA polymerase stays put for a bit while the first 9 base pairs are added, then elongation will occur

• Nucleoside triphosphates (NTPs) are added one by one

Elongation…

• just adding more NTPs, about 12 bp are bound to DNA as it goes

• DNA rewinds behind RNA polymerase

• elongation occurs from the 5’ to 3’ end

Termination

• terminator sequence at the end of the transcriptional unit, which contains a sequence of adenosines that code for uracil’s, and then a GC rich region

  • Note: the terminator is in the secondary structure of the transcript, NOT the primary sequence of the DNA

• GCs led to the formation of a hairpin loop

• hairpin loops mostly but also a little uracil’s signal transcription to end

We have 3 kinds of RNA polymerases…

RNA polymerase II is responsible for making pre-mRNA (In eukaryotes)

initiator sequence is…

the sequence surrounding the start point

• upstream from that is the TATA box

• TATA box + initiator = core promotor that exists on every gene in the genome

Transcription factors

class of proteins that aid in transcription, they will be activated and then bind to the promotor as well, this will help RNA pol II to bind

What are basal transcription factors?

transcription factors that don’t need to be activated, and are required for RNA pol II to bind to DNA

• for RNApol II TFIID (TF2D) is it and it binds to the TATA box

• more transcription factors bind, and the more that do the more transcription is promoted even more, this complex they make is called the pre-initiation complex

• then RNA pol II binds

What are some medications that occur to the primary transcript mRNA after transcription?

• 5’ cap

• poly(A) tail

• splicing

we need these because mRNA is incredible unstable, it degrades VERY easily, these help to stabilize it a bit

5’ Methylated cap (backwards cap, 5’ to 5’)

methylated guanine added during elongation, soon after the first nucleotides are places

Functions:

• flags the mRNA for nuclear export into the cytoplasm

• protects against degradation from exoribonucleases

• binding site for the ribosome during translation

it is added on by a capping enzyme that only associates with RNApol II, meaning it only adds to mRNA

Polyadenylation

(goes on the 3’ end)

transcripts are generally too long, so we need to mark the ends which is which

• poly(A) polymerase or PAP finds the poly(A) signal of AAUAAA that marks the end of the important stuff, cleaves the rest off and adds in a poly(A) tail

• poly(A) tail protects the 3’ end, can recruit proteins

Functions: important for translation, enables it just like 5’ cap, and protects the 3’ end

modifications only last for so long…

• slow degradation of poly(A) binding proteins, and a de-capping protein that released off the 5’ cap

• therefore, mRNA does not equal protein

exoribonucleases are important for…

protecting us against ribosomal bacteria

exons usually represent protein domains

mRNA contains both the introns and exons of the DNA, this is the pre-mRNA, and splicing, we have mature mRNA which is used for translation

Splicing…

typically removes introns

• numbering of primary transcript skips over the introns, after splicing we have a continuous mature RNA

• first produced mRNA is a pre-mRNA, or primary transcript

• splicing produces a mature RNA, with a poly(A) tail and a 5’ cap

Splice sites and the spliceosome…

• marked consensus sequences called splice sites mark the beginning and end of introns

• GU on 5’ end and AG on 3’ end (100% conserved)

• catalyze the formation of the spliceosome which mediates splicing, it is made up of proteins and RNAs (snRNPs or small nuclear ribonucleic particles)

Splicing reaction…

• splice sites are where snRNPs will come in

  • U1 binds to GU

  • U2 binds to branch point sequence a bit before AG

  • U4, U5, and U6 come in to bind

• 5’ end is broken off and rebound to the branch point sequence creating a lariat structure

• cue to cleave 3’ end, combine the exons and remove intron

• intron without protection on its ends will degrade

Gene expression is controlled primarily by…

transcription

Anabolic steroids….

act by modifying gene regulation, to build something up

How are genes regulated within prokaryotes?`

operon model:

• operon: set of genes that have a related function with one another, that are all encoded together in one mRNA strand, and then synthesized together to produce their proteins, this means there should be a 1:1 ratio of these because they’re transcribed together

• polycistronic mRNA: mRNA that codes for more than one polypeptide, this is only found in prokaryotes, eukaryotes are monocistronic

Explain gene regulation for the lac operon…

Galactoside permease: shuttles lactose into the cell

Beta-galactosidase: catabolizes lactose into galactose and glucose

• both of these are encoded together on the lac operon

• active lac repressor binds to the operator and blocks transcription, when lactose is available, it binds to the lac repressor and inactivates it so it can’t bind to the lac operon, which allows transcription

Operons consist of…

• structural genes: code for the enzymes/proteins of interest

• regulatory genes: control the expression of structural genes by expressing regulatory proteins (operator)

Operator…

• sequence located between the core promotor and first structural gene

• proteins may bind here and promote or inhibit transcription(repressors)

Repressors…

• bind to the operator and inhibit the operon (when it is active)

• when the repressor is altered, aka allosteric regulation by the effector(substrate)

• physically blocks RNA polymerase from binding to the promotor

• negative control of transcription, because binding of a protein to the operator turns transcription off

• substrate induction: substrate induces its enzyme (allosteric reg to the repressor, ex: lac)

explain regulation for the trp operon…

• in the presence of tryptophan, the repressor for the trp operon binds to the operator, repressing it

• end produce repression what is produced by the operon ends up leading to its own repression

Positive control of the lac operon…

• glucose pathways are constitutively expressed(always on) because we must have energy to make ATP

• in the presence of glucose, alternative pathways such as the lactose pathways aren’t turned on

• in the lack of glucose, these pathways are upregulated more so that glucose can be produced

• if glycose is low, cAMP will be high so it can form a complex with CRP (catabolite regulation protein) that serves as a transcription factor required for polymerase binding to transcribe the lac operon

Explain terms related to transcription of the eukaryotic DNA…

cis-acting elements: regions in/around the promotor to which regulatory proteins may bind (same chromosome)

trans-acting elements: regulatory genes that exist far away on the DNA

• ex: the gene for a repressor

• the products of a trans-acting elements, trans acting factors, are proteins that bind to cis-acting elements

trans-acting proteins (transcription factors) come and bind to cis-acting elements (DNA sequences)

• transcription factors can act on many different cis-acting elements on completely different genes, additionally, for a single gene, there are multiple different transcription factors that can bind to that gene

More transcription factors…

bound to a gene = increased transcription, more mRNA copies, more readily recruits RNA pol II

Types of Cis-acting elements…

Proximal control elements: < 100 bp up or downstream from the promotor

distal control elements: > 100 bp up or downstream from the promotor (typically 1000s)

• enhancers: folding of chromosomes can cause 1000 bp away to actually be close to a promotor

• repressors

MODY(diabetes) and HNF

HNF: Hepatic nuclear factor, a transcription factor that only binds to DNA when its(HNF) in a dimer form

DCoH: dimerization cofactor of homeodomains, a protein required for certain transcription factors to dimerize and associate with the transcriptional machinery

• HNF requires DCoH to function in the pre-initiation complex, and DCoH requires HNF to bind to DNA, they must become a heterotetramer so stable HNF can bind to DNA and promote the production of insulin

• mutations of HNF inhibit this interaction

Treatments for MODY…

• insulin

• oral hypoglycemics

not quite there yet…

• pancreatic islet transplant

• pancreas transplant

Steroids…

• every steroid has a similar structure, they’re incredibly hydrophobic so they can transport through cell membranes easily, but need to be transported through the blood via albumin

• use intracellular receptors: when it binds, it promotes the release of inhibitor proteins that now act as transcription factors and promote transcription

EX; anadrol

• binds to androgen receptor (AR) more strongly and more specifically than natural testosterone, so it doesn’t let go, keep expressing the proteins

• prevents some post-translational modifications of the receptor

• AR receptor promotes insulin-like growth factor (IGF-1) which promotes protein biosynthesis and cell growth, and inhibits pathways that promote protein degradation

Details about the genetic code…

• consists of a triplet code, aka 3 bases code for a particular amino acid, those 3 bases are called a codon

• 64 possible combinations, however multiple codons can code for a single amino acid

Features vocab:

• degenerate: an amino acid can be coded for by more than one codon

• unambiguous: each codon indicates a single, specific amino acid

• non-overlapping: when translated, the “reading frame” is advanced 3 bases at a time

• 61 codons encode for amino acids, the remaining 3 are stop codons that terminate the polypeptide

Which codon is the start codon?

AUG - methionine

stops are …UAA, UAG, UGA

tRNA or transfer RNA…

• specific to each codon

• anticodon: is antiparallel and complementary to the codon it is for

• acceptor stem: where the amino acid its shuttling in is attached, joined by an ester bond

• 3 hairpin loops, one with the anticodon, the other 2 are for stabilization, help it interact with ribosomes

While we have 61 codons, there are not 61 tRNAs

Wobble position: the 1st position of the anticodon, meaning what binds to the 3rd position of the codon does not have strict binding, this causes multiple codons to be recognized by one tRNA

• A in anticodon will bind to U in codon

• C in anticodon will bind to G in codon

• U in anticodon will bind to A or G in codon

• G in anticodon will bind to C or U in codon

• I(inosine) will bind to U,C, or A in codon

Inosine

• only found in tRNA, typically in the wobble position

• can bind to U, C, or A

• it arises by deamination of adenine

Aminoacyl-tRNA Tranferases

• links amino acids to the correct tRNA to load it

• one transferase per amino acid

• requires 1 ATP to form the ester bond

• the free energy of that bond is used to form the peptide bond between adjacent amino acids in a growing polypeptide

mRNA from the ribosome’s POV

• 5’ untranslated region (UTR): everything that is before the start codon

• 3’ UTR: everything after the stop codon [help contribute to the kinetics of translation]

• coding (DNA) sequences (CDS): everything from and including the start codon up until the stop codon [post transcriptional modifications]

They provide key regulatory feedback

Ribosome…

• 2 subunits, large and small

• composed of ribosomal protein and ribosomal RNA (rRNA)

Prokaryotic vs. Eukaryotic ribosomes

Prokaryotic:

• 50S and 30S subunit sizes (Svedberg units, how they pull out when spun)

Eukaryotic:

• 60S and 40S

Ribosomal binding sites:

• mRNA binding site

• A site (activated tRNA enters with an amino acid)

• P site (growing polypeptide is attached to the amino acid on the tRNA)

• E site (empty tRNA is present, exits the ribosome)

continuous process

Initiation in prokaryotes…

• initiation factors (IFs) help the small subunit to sock to the ribosome binding site (Shine-Dalgarno consensus sequence)

• Formyl-methionine (fMet) binds to the start codon

• energy from GTP bound to an initiation factor is used to complete assembly of the ribosome

Initiation in eukaryotes…

• Methionine, not fMet

• Eukaryotic initiation factors (eIFs)

• no consensus sequence for ribosome binding site

• “Forward search”: complex of eIF2-GTP and tRNA(met) binds to 5’ cap and searches forward for the first AUG, then the large subunit comes in and binds via another GTP hydrolysis

Elongation…

1. binding of tRNA, elongation factor proteins(EF-Tu, serves as a shuttle, has 2 GTPs) that brings the next tRNA to the A site(if the tRNA is wrong it wont do the rest)

2. peptide bond formation from energy from ester bond (peptidyl transferase)

3. translocation, another elongation factor(EF-G) pushes everything down one base

Termination…

• when stop codon arrives in the A site

• a release factor binds in the A site, not a charged tRNA

• the peptide chain transfers to a water instead of to an amino acid, creates a carboxy terminus, release of peptide chain

• translational complex falls apart

Other notes:

• folding proceeds as the peptide chain is synthesized

• 1 ATP and 3 GTPS are used for each amino acid added

• Many ribosomes proceed along a single mRNA in a chain, polyribosomes, which maximize efficiency

• polycistronic mRNA, can contain many multiple ribosome binding sites, one for each protein in the operon, allowing simultaneous but separate translation of all encoded proteins (prokaryotes only)

• some cellular mRNAs and many RNA viruses contain an internal ribosome entry site (IRES) that enables translation to occur in a cep ended manner

DNA substitutions by mutation or variation

• Missense: a base change leads to a change of amino acid

• Nonsense: a base change leads to a stop codon

• Silent: a base change has no effect on the amino acid sequence

DNA insertions and deletions…

• Frameshift: a base is inserted or deleted from the sequence, leading to a shift in the reading frame of the ribosome

• definite missense, possible early stop codon