biochem exam 5 (34-41)

2) Elongation

- Bound holoenzyme isomerizes from a closed to open complex that contains unwound DNA. - The transcription bubble is the region of RNAP unwound DNA that includes the RNA-DNA hybrid. - Productive elongation requires release of s. - RNAP elongation is slower than DNA polymerase and more error prone. "the template strand is what's used to make RNA." ????????????? recheck slides from beginning of lecture//confirm with dr wold??? "once initiation has occurred, isomerization from closed to open open complex and now made this "bubble""

two types of complexes and type of modification

- Chromatin modifying complexes - make post-translational modifications "like small change like painting a house" - Chromatin remodeling complexes - change position of nucleosomes ";ike big change like removing a wall of a house"

Maturation of the mRNA: splicing (many parts) splicing of pre-mRNA: signals

- Cis-acting elements in mRNA: "within DNA, on the element itself" - Splice junction: most common 5'- GU, 3'- AG, but not invariant. - Branchpoint sequence, A: 20-50 nt from 3' splice site - Polypyrimidine tract: just upstream of 3' splice site - Trans-acting "must go to DNA, works ON the element, usually reads out the element" - Spliceosome: a complex of 5 small nuclear (sn) RNAs plus proteins (snRNPs): Named after the snRNA - U1, U2, U4, U5, U6 snRNP

tRNA recognition by Aminoacyl tRNA-synthetases

- Crystalstructuresoffive tRNA- synthetase pairs. - tRNAs(red)havea similar shape, but how and where interact with a synthetase differs. - Recognitionrequires interactions with multiple regions, NOT just the anticodon. "red=tRNA location/interaction "here is an example of 5 diff synthetases shown"

Maturation of the mRNA: 5' capping

- Modification of a 7-methylguanylate attached by a 5' to 5' linkage between a ribose and a triphosphate at the 5' end of the mRNA. - Occurs co-transcriptionally - Involves: - Removal of Pi from 5' triphosphate - Attack of GTP at the a-phosphate to form a 5' to 5' linkage. - Methylation of the N7 of guanine and the ribose sugar of the two adjacent bases. - Needed for eukaryotic translation initiation.

E. coli RNA polymerase and its subunits there are 2 primary forms of RNA pol

- Multi-subunit enzyme: - Initiating holoenzyme: a2bb'w plus s (alpha, alpha 2, beta, omega AND sigma) - Elongating core enzyme is a2bb'w ((alpha, alpha 2, beta, omega WITHOUT sigma)

non-AA codons three stop codons? exceptions for Mer, Trp exceptions for Arg, Ser, Leu

- Not all codons specify AAs.- Three stop codons: UAA, UAG, UGA - The number of codons for each AA ranges from 1 to 6: - Met, Trp: 1 codon; Arg, Ser, Leu: 6 codons "if there is no corresponding AA, it could be a stop codon and those are recognized by release factors ie: transcription factors. the stop codons are not recognized by tRNA"

Maturation of the mRNA: 3' polyadenylation

- Occurs co-transcriptionally. - Stimulates RNA Pol II release .- Requires a polyadenylation signal (poly-A) AAUAAA, endonuclease called the cleavage and polyadenylation specificity factor (CPSF) and poly(A) polymerase. - Increases RNA stability and improves translation.

Alternative splicing: more a rule than exception

- One gene can produce multiple mRNAs with different exon combinations, producing multiple proteins. - About95%ofhumangenesarealternativelyspliced. look at fund genetics for notes on this

tRNA selection by synthetase: what is needed?

- Recognize the anti-codon loop and acceptor stem, plus other features. - Modified bases can be discriminating components. - Distinct for each synthetase.

CTD phosphorylation of which Ser leads to what outcome? ser 5 ser 2 ser2/5

- Ser 5: Capping machinery - Ser 2: Poly A machinery - Ser 2/5: Splicing

Principles of alternative splicing

- Splicing choices depend on the relative strength of splice sites, with a stronger consensus spliced well. - Regulated splice sites have weak consensus sequences and are controlled by cis-elements called exonic or intronic splicing enhancers (ESE, ISE) and exonic or intronic splicing silencers (ESS, ISS).

sigma of holoenzyme

- binds the -10/ -35 elements in a promoter. - is released when the new RNA chain is ~9-10 nucleotides. There are multiple sigmas in E. coli, with sigma abundance dependent on growth conditions. The major sigma is s70.

1. Comparepropertie sof Prokaryotic RNA Polymerase and Eukaryotic RNA Pol II: Eukaryotes Prokaryotes Need for a primer? Synthesizes tRNA? Promoter sequence? Promoter recognition?

- euk and prok don't need a primer (DNA pol needs it to start but RNA can use transcription bubble" prokaryotes have RNAP that synthesizes all RNA types, euk RNAPII doesn't do tRNA but does mRNA promotor sequence for euk is TATA box for RNAPII, for prokaryotes it is -10/-35 upstream of transcription promotor recognition? RNAPII doesn't recognize promotor, TFIID does and its not a part of RNAPII but just associated with it? prok RNAP does recognize promotor bc the holoenzyme is a RNAP with the sigma factor

prokaryotic versus eukaryotic replication

E. coli Small genome (4.6 x 106 bp) -Single circular chromosome -One origin -Doubles every 20-30' -DNA Pol III/ I -Primase -SSB Human Large genome (6 x 109 bp) -23 pairs of linear chromosomes -Tens of thousands of origins -Replicates in 8 hrs -DNA polymerase (delta/ epsilon) -DNA polymerase alpha -Replication Protein A: RPA

the lac operon what does it encode for? what is it controlled by?

Encodes three proteins: - lacZ: b-galactosidase (involved in lactose breakdown into glucose and galactose ) lacY: permease lacA: thiogalactoside transacetylase (transacetylase) Is controlled by: - Cis-acting elements: (elements in DNA itself) Promoter Operator Catabolite activator protein (CAP) binding site - Trans-acting factors: (proteins that will interact w DNA) RNAP, Lac repressor (lacI), CAP

Pol II regulation regulation: enhancers

Enhancers: - Are long-distance acting elements. - Bind transcription factors (activators), often in a tissue- specific fashion. - Activate transcription in a position- and orientation- independent fashion.

How is Rho dependent transcription termination different from rho independent termination?

In Rho-dependent termination, a Rho protein is responsible for transcriptional termination. The formation of the hairpin loop structure, on the other hand, results in Rho-independent termination. Therefore, this is the main difference between Rho-dependent and Rho-Independent termination.

Chapter 36: RNA Synthesis and Regulation in Bacteria Outline: Types of RNA and maturation RNA polymerase and transcription stages Prokaryotic gene structure Regulation of lac, trp operons

Key Concepts • Structure and function of RNAP. • Gene structure: promoter, operator, terminator • Basal versus activated transcription .• Regulation of the lac operon .• Regulation of the trp operon.

Regulation of the lac operon when does repress occur

Lac repressor (lacl): - Binds the operator - Blocks RNAP binding to promoter if there's no lactose, it is repressed. no need to make enzymes that breakdown lactose I =f lactose is not present. "lac repressor (lacL) is a block, meaning the polymerase can't bind and transcribe this set of genes bc the genes are not needed when lactose isn't there to be broken down"

lecture 34-Chapter 34: DNA replication Outline: • Seven enzymatic activities for replication • Origin recognition protein • Mechanism of replication • Specificity and fidelity • Coordination of replication of two strands • Replication of linear chromosomes See problems #1-6, 9, 10, 12, 17

Lecture 34: Key Concepts - Names of seven activities required for DNA replication. - Define how replication fidelity is achieved. - Define differences between leading and lagging strands. - List shared and distinct properties of DNA Polymerase I and III. Define how these Polymerases are used in replication. - Define the structure of the replication origin in E. coli, and the replication fork. - Explain telomere structure and enzymes involved in formation. - Explain the reason telomerase is needed in eukaryotes but not prokaryotes.

Chapter 37 Gene Expression in Eukaryotes Outline: - Three RNAPs- RNAPII Regulation - Cis: promoters, enhancers- Trans: GTFs, Mediator, HATs, chromatin modifying complexes- Nucleosomes and transcription

Lecture 37: Key Concepts • Function of three RNA polymerases• Structure of RNA Pol II regulated gene: promoter elements, enhancers. • Role of the RNA Pol II CTD. • Function of the basal transcription factors: TBP, TAFs, Mediator. • Histone and nucleosome structure.• Effects of histone modification and DNA methylation on transcription.• Types of TFs: chromatin remodelers and modifiers.

Chapter 38: RNA Processing in Eukaryotes Outline: - rRNA, tRNA processing - mRNA maturation: - capping, splicing, polyadenylation - coordination of processing - Self-splicing RNAsSee problems #1-9, 12-14, 16, 17 at the end of Chapter 38.

Lecture 38: Key Concepts • Maturation of rRNA: snoRNPs • Maturation of tRNAs: RNAse P, nucleotidyl transferase, base modifications • Maturation of mRNAs: capping, polyadenylation, splicing • Mechanism of splicing: cis-signals and trans-acting snRNPs • Alternative splicing • RNA as a catalyst • Contributions of splicing errors to human disease

Chapter 39: The Genetic Code Outline: - Rules of the genetic code - Critical components in code translation - tRNA structure - AA-tRNA synthetases - Ribosomes

Lecture 39: Key Concepts • Know the features of the genetic code .• Know the structural properties of tRNAs. Understand how to write the sequence of a tRNA anticodon. • Understand the function of Aminoacyl tRNA synthetases, including how tRNAs are recognized and correct charging of amino acids is accomplished. • Be able to use the codon table to translate a mRNA sequence. • Understand the principle of wobble.

DNA ligase reactions

Ligase- formation of new phosphodiester bond joins the free 3'OH of one base with the free 5'phosphate group of one base ATP dependent reaction this reaction is important for coordination with leading and lagging strands

anticodon loop

One of three loops on a tRNA which contains the 3 nucleotides that allow it to align specifically with mRNA. Contains the anticodon

three eurkaryotic polymerases and promotor types "diff DNA sequences that GTFs bind to to bring in the diff RNA pol"

Pol I: Makes rRNA> 100 tandem copies of same gene Promoter elements close to transcription start Pol II: Makes mRNAs, snRNAs Many different genes Promoters have interchangeable elements Pol III: Makes tRNA, 5S RNA Promoter elements are downstream of start Promoter fall into two classes "only downstream promotor is for RNA pol III" remember, transcription starts at +1, upstream/downstream is in relation to this

E. Coli DNA polymerases: classis (replicative) polymerases

Polymerases I & III have 5'-3' polymerase activity "join units of nucleotides" have 3'-5' exonuclease activity (proofreading) are processive enzymes (make long stretches of DNA without dissociation) displays fast rates of synthesis posess high fidelity (make one mistake every 10^9/10^10 nucleotides)---"usually very accurate in incorporation"

Gene expression: prokaryotes vs eukaryotes

Prokaryotes - Single RNAP (transcribes all three RNA types" - Promoters recognized directly by RNAP "sigma factor" - Repressors and activators directly affect RNAP "ex: lac repressor, CAP, trp repressor" - Transcribed mRNA is mostly in mature form - Coupled transcription and translation "trp operon is an ex of this big difference, bc the nucleus in eukaryotes have compartmentalization of translation and transcription so they aren't coupled" Eukaryotes - Three RNAPs - Promoters recognized by general transcription factors (GTFs) that recruit RNAP "recognized bt TFIIH which isn't in RNAP but alongside it", "similar to CAP in that they help recruit RNAP" - Repressors and activators affect DNA accessibility through effects on nucleosomes - Transcribed mRNA is mostly processed - Nuclear transcription, cytoplasmic translation "not coupled/separated"

- Cis-acting elements: (elements in DNA itself)

Promoter Operator Catabolite activator protein (CAP) binding site

the lagoon strand is in fragmented pieces: Okazaki fragments

- primase initiates RNA synthesis at multiple positions on the lagging strand to make RNA primers of ~10 nucleotides - DNA Pol III extends RNA primers, forming short DNAs called Okazaki fragments - the 5' to 3' exonuclease activity of DNA pol 1 removed the primer DNA, synthesizing DNA to fill the gap. DNA pol III cannot remove a primer since it lacks 5'-3' exonuclease activity - DNA ligase catalyzes the formation of a phosphodiester bond between Okazaki fragments

tRNA (transfer RNA) maturation

- tRNA processing involves: - 5' leader removal by RNAse P (an RNA containing endonuclease) - 3' end trimming by an endonuclease, RNase Z - Addition of CCA at 3' end by tRNA nucleotidyltransferase- Intron removal (in a minority of tRNAs) - Base modifications, for example pseudouridine, average ~13 modifications per tRNA

DNA pol I and III insight

1) RNA primase lays down an RNA primer 2) DNA POl III extends the RNA primer 3) DNA Pol I removes the primer and replaces it with DNA 4) DNA ligase forms. bond joining the two DNA fragments

Splicing of pre-mRNA: two basic principles

1) RNAs play direct role in alignment of splice sites and in catalysis. 2) Energy required for splicing used by ATP-dependent helicases to unwind RNA duplexes intermediates to facilitate catalysis.

types pf RNA transcription produces

3 major classes but there are more than this messenger RNA - most variant types, give rise to protein synthesis mRNA molecules carry the genetic information needed to make proteins. They carry the information from the DNA in the nucleus of the cell to the cytoplasm where the proteins are made ribosomal RNA- most abundant type of RNA,help w translation help translate the information in messenger RNA (mRNA) into protein. transfer RNA- Transfer RNA serves as a link (or adaptor) between the messenger RNA (mRNA) molecule and the growing chain of amino acids that make up a protein.

translation has a direction, mRNA reads ____?

5'-3' so mRNA cide position 3 is tRNA position 1

Branchpoint sequence, A

: 20-50 nt from 3' splice site

Spliceosome

: a complex of 5 small nuclear (sn) RNAs plus proteins (snRNPs): Named after the snRNA - U1, U2, U4, U5, U6 snRNP

Splice junction

: most common 5'- GU, 3'- AG, but not invariant.

base modifications in tRNA

???

CTD phosphorylation cycle

CTD phosphorylation promotes recruitment of protein complexes involved in mRNA maturation "ex: need capping for mRNA maturation" "when Ser 5 of CTD is phosphorylated then it recruits the "capping enzyme". which adds a cap to the mRNA being transcribed, which allows the mRNA to be translated. after more phosphorylation and phosphorylation of Ser2, can recruit specific splicing machinery. this is some of the final steps in the maturation of mRNA"

activation of CAP to help BASAL level of transcription of the lac operon

Catabolite activator protein (CAP): - Binds DNA when cAMP is present (low or no glucose) - Improves recruitment of RNAP to promoter - CAP-cAMP forms protein-protein contacts with RNAP - Stimulates high levels of transcription: ACTIVATED "CAP is also a guy who is a DNA binding protein, it will bind to sequences upset of the -10/-35 and will. help RNA Pol say yes to -10/-35 and transcribe the gene. if CAP is bound to cAMP, DNA binding will occur and it will bind to sequences upstream of the -35 and help RNA polymerase identify the -10/-35 promotor region. Cap only binds when cAMP is present. when cAMP is high and glucose is low, int he presence of lactose, you have CAP binding to the promotor region, helping DNA pol bind, with no roadblock, you have lots of synthesis and now start making B-galactosidase and we now have high levels of transcription"

Self-splicing RNA: No protein needed (Iowa City rom tech finding)

Catalytic RNAs discovered in the ciliate Tetrahymena. Involved in removal of an intron in rRNA. - Categorized as Group I introns. - Requires an added guanine that acts as a nucleophile to start the splicing reaction. - Guanosine is transiently incorporated into RNA.- The 3' OH of the free exon attacks the 3' splice site, joining exons

DNA compaction

DNA compaction is needed for eukaryotic genomes to fit inside the nucleus. - Compaction of ~7X achieved using nucleosomes composed of histones. - Interactions between nucleosomes and higher order folding establish additional levels of chromatin folding.

1. Transcription Initiation: what is the role of the promoters

Promoters: - Bind RNA polymerase (RNAP) holoenzyme- a2bb'ws. - Share common sequence: -10, -35 consensus. - Are recognized by the omega subunit (-10/-35) and the alpha subunit of the core enzyme (UP). - Strength (efficiency) correlates with match to the -10/ -35 consensus sequence and presence of UP. - Usage is regulated by transcription factors. "promoter is the region where RNA pol is going to bind 2 ways to have promoter identified: -the alpha in UP subunit and sigma in holoenzyme AT rich is about 40/60 upstream and it is called the UP- element, helps recruit RNA Pol - sigma from holoenzyme typically binds to the region of 10/35 called the consensus."

Attachment of an AA to a tRNA: getting it right what is linking the AA to tRNA how is specificity ensured?

Synthetases link the AA to the tRNA: usually one per AA. Specificity for an AA is ensured by: - geometry of active site (acylation site) - geometry of editing site

Pol II regulation

The Carboxyl Terminal Domain (CTD): - is found on largest subunit RNA Pol II - coordinates functions of RNA Pol II- contains repeats of (YSPTSPS)n - is hypo-phosphorylated during INITIATION - is hyper-phosphorylated during ELONGATION - shows ordered phosphorylation that permits recruitment of mRNA processing factors: Ser 5 then Ser2 "coordinates function important to make RNAPII start transcribing and bring machinery that going to process and make that RNA so it can be translated"

functions of the lac operon

Purpose: - Breakdown lactose if glucose is not available. - b-galactosidase cleaves lactose into glucose and galactose. Induction of the operon increases levels of b- galactosidase from 10 to ~1,000 molecules per cell. - Permease transports lactose into the cell. - Transacetylase detoxifies collateral compounds imported by permease. "if lactose is added, b-galactosidase will increase to breakdown lactose to glucose and galactose for energy use?"-graph interpretation fund genetics: If the operon makes enzyme that breaks down lactose, when lactose is not present there is no point in making the enzyme No lactose --> turn off the lac operon-- lac repressor (lacl) Back to the idea of conservation of energy, why create an enzyme that doesn't have a substarate to work on?

RNA Pol II promotor types?

RNAP II promotor contains a TATA box, initiator (Int), and a downstream promotor element (DPE) "diff genes can have diff usage of the promotor elements some can have only TATA/initiator some can have initiator/DPE this is due to the flexibility of th TF's"

- Trans-acting factors: (proteins that will interact w DNA)

RNAP, Lac repressor (lacI), - down-regulation CAP- up regulation

Regulation of the trp operon: Attenuation (inhibition)

Regulation depends upon: - Coupling translation of leader RNA sequence with RNA polymerase transcription. Structures formed by the leader RNA. "what determines the structure of RNA that's coming on or off is the ribosome. in absence of trp: ribosome sits and waits for tryptophan tRNA but because its been waiting and RNA pol is going, it makes a structure that doesn't include termination In presence of trp: we have trp and now RNA pol is charging through, and so is the ribosome. the ribosome thinks "I have tryptophan, I can keep moving", so it moves along and what happens is you start to make a 3/4 stem-loop hybrid and now you make the termination signal. make a small RNA and that small RNA DOES NOT encode the synthesis enzyme of tryptophan so we don't make them. attenuation protects RNA pol from going down the entire gene and control level of biosynthetic molecules

3) Protein-dependent termination

Rho (r): - is a hexameric protein with translocase (ATPase) activity - binds a recognition sequence in RNA - uses energy of ATP to translocate on RNA towards RNAP - has an inside channel where RNA passes - breaks the RNA/DNA hybrid in the transcription bubble "Rho is a termination factor, it also has to recognize a sequence and that that sequence it binds to RNA itself. it uses ATP hydrolysis to move, when it gets to RNA/DNA hybrid, it will cause an unwinding and a release of the RNA. uses energy of ATP to translocate, it pulls the RNA though that inside channel like we saw before with helices and it will break that RNA.DNA consequently causing RNA polymerase to terminate

Readers of the code: tRNAs

Shared features:- Length: 73- 93 nucleotides - Shape: -2D-cloverleafwith5unpairedregions-3'CCA, TyC,Darm (dihydrouracil), the extra arm, anticodon loop - 3D- L-shaped with AA and anticodon on opposite ends. - Composition: contain unusual bases that promote synthetase identification, base pairing and stability.

Antibiotics target transcription

Two common antibiotics target transcription: - Rifampicin: binds RNAP, blocks transcription after a few nucleotides. Binds the RNAP b subunit, prevents elongation. Inhibits only prokaryotic RNAPs. "similar to if there was no sigma dissociation" - Actinomycin binds DNA in the transcription bubble and blocks elongation. Inhibits prokaryotic and eukaryotic RNAPs. High concentrations will affect DNA polymerase.

2 mechanisms of regulation of trp operon

Two mechanism regulate production of trp mRNA: - Repression: repressor bound to Tryptophan binds DNA - Attenuation: early transcription termination regulated by translation. "this one has a different principal it illustrates. that is that the E.coli has no nucleus, as soon as transcription is going and mRNA is being produced, translation machinery is coming up to mRNA and making protein. so, transcription and translation are coupled. because that is true, , the cell takes advantage of such coupling for regulation of transcription. so the coupling of transcription and translation is used to regulate the production of some of the biosynthetic machinery. that is important for translation. what is that machinery? well, the trp operon encodes for enzymes to make tryptophan. tryptophan is a required amino acid for protein synthesis. the more tryptophan we have means we don't need to make more of these enzymes because ira a waste of energy so you wanna regulate the operon. "

a tRNA is named based units anticodon. tRNAtrp or tRNAcca that reads codon?

UGG

histone tails can undergo types of modification major two types to know?

acetylation (activation) methylation (activation or repression) they change the access to DNA histone tail modifications alter transcription

histone acetyl transferases (HATs)

add acetyl groups

tRNA Nucleotidyltransferase Enzyme

addition of CCA at 3' end " when AA is attached"?

RNAse Z

an endonuclease that removes the 3' end on tRNA

RNAse P

an endonuclease that removes the 5' leader on tRNA

Coactivators

are proteins that interact with general transcription factors, RNA polymerase or nucleosomes to modulate transcription. "mediator is a type of this:

A portion of the nucleotide sequence of the template strand of DNA from E. coli is shown below. This sequence is known to encode the carboxyl terminus of a long protein. Determine the encoded amino acid sequence. 5' ACCGATTACTTTGCATGG-3'

ask arshi bc idk

You are in a biochemistry lab and make mutations of the lac operon. Predict the likely effects of mutations that: 1. Move the operator to downstream of the transacetylase gene. 2. Make a promoter mutation where the catabolite activator binding site is deleted.

ask wold

TBP-associated factors (TAFs)

bind the Inr (initiator) and DPE in TATA-less promoters.

TBP (TATA binding protein

binds the TATA box. "bind in the minor groove of DNA which doesn't happen as often, this leads to a really distorted promotor region, essentially bending DNA"

Anticodon on tRNA direction of anticodon direction of mRNA complementary to anticodon

binds to the mRNA in a complementary fashion

TFIIH

unwinds the promoter (helicase) and phosphorylates Pol II CTD (kinase) at Ser5 "when TFIIH is recruited, it has two helpful properties for transcription, it 1) contains a subunit that's a helicase (uses ATP-energy), and that's gonna help make the transcription bubble, help the open complex, the open complex to help identify the template strand to start RNA synthesis. the 2) is it contains a kinase that phosphorylates the CTD ((carboxyl terminal domain) to begin to allow Pol II to get its properties it needs to start elongation. so, go from open to closed, o the open the complex has NTPs? (nucleotides?)that come in initiationlyy transcribing and then you have an elongation-complex"

how are most E.Coli genes arranged what do these contain

usually arranged in operons (within DNA) Operons: - contain two or more structural genes (protein coding) - are controlled by a single regulatory region, with a single promoter and operator. - produce a single mRNA. - allow coordinate gene expression.

defects in helicase can cause what? what's an example

can cause human disease an example is Werner syndrome caused by the mutation of WRN (RECQL2) gene that encodes 3'-5' DNA helicase disrupts hydrogen bonds

3' CCA

conserved sequence at the 3' end of all mature tRNA molecules that functions as the site of amino acid attachment

prokaryotic versus eukaryotic RNAP structures

contain beta prime, beta, alpha 1, alpha 2, omega (similar to both euk and prok) euk RNAP contain common subunits as well specified by their additional enzyme-specific subunits: RNAP I has +5 RNAP II has +3 RNAP III has +7 (most diverse)

coordination of leading and lagging strands is due to what?

coordination occurs due to dimerization of the polymerase the lagging strand loops out to form a structure that acts as a troone slide does, growing as the replication fork moves forward when lagging strand polymerase reaches a replicated region, the sliding clamp is released and a new loop forms

A mRNA was isolated from mitochondria and translated using a cytoplasmic protein extract. Surprisingly, the size of the translated protein did not match the known size of the mitochondrial protein. In fact, it was smaller than expected. Propose a hypothesis for this difference. What principle of the genetic code does this finding illustrate?

cytoplasmic has UGA stop codon that mitochondria doesn't have. this should lead us to start thinking: smaller protein being produced means there was a stop codon that was read too fast. we looked back and found a stop codon present in the cytoplasmic that wasn't found in the mitochondria remember that mitochondria has its own genome and tRNAs which results in mitochondria having a slightly different code despite being an organelle within cells so weird that mitochondria is created by a a cell that has an entire genome and its creates mitochondria that has a different entire genome????

Variants of splice sites contribute to human disease

ex: sensitivity to covid 2'-5' olgioadenylate synthetase 1 (OAS1)

operon

fund genetics: operons are within DNA ribosome can initiation protein synthesis from internal sites in the mRNA--> more than 1 protein is formed you can have. Single promoter that creates a single rna that encodes for deveral genes at the same time. genes in an operon are coordinately controlled, one mRNA simultaneously translate more than one protein

telomerase

fundamental genetics: Telomerase carries a short RNA molecule that acts as a template for the addition of DNA at the 3' end This is an example of reverse transcription (RNA-->DNA) -is responsible for forming chromosome ends, telomeres. -has a protein with an RNA component. -uses a 3' OH of single strand DNA overhang as a primer and RNA as a template to synthesis telomeric DNA, a GT rich repeat. Multiple copies of the repeat are added. -is known as a cellular reverse transcriptase (reverse transcription bc going to RNA to DNA.

role of helicase where it gets its energy ? structure? how does it change DNA?

helicase separate DNA strands helices use the energy of ATP hydrolysis to separate strands of the double helix to make DNA available for DNA polymerase helicase usually has 6 subunits that form a ring-like structure around DNA. each subunit has a loop that interacts with DNA ATP hydrolysis by helicase subunits causes conformational change that pulls DNA through the helicase center

factors at the replication fork

helices- unwind DNA SSBs- prevent secondary structure od DNA from forming topoisomerases- releive stress due to unwinding of DNA primase- makes the RNA primer polymerases- add nucleotides5'-3' *one strand is continuous: leading strand: DNA Pol III * one strand is discontinuous: lagging strand: DNA Pol I and III nucleases- DNA Pol I (RNA, editing) and Pol III (editing) ligase- lagging strand

roles of primary forms of RNA pol

holoenzyme- initiating enzyme,involved in regulation core- elongating enzyme it transcribes all RNAs: mRNA, rRNA, tRNA

elongation effect on phosphorylation of carboxyl terminal domain (CTD)?

hyper-phosphorylation

initiation effect on phosphorylation of carboxyl terminal domain (CTD)?

hypo-phosphorylation

chapter 35

in pharmacology final exam quilt :/ too lazy to transfer here

- lacZ: b-galactosidase

involved in lactose breakdown into glucose and galactose

transcription bubble

is the region of RNAP unwound DNA that includes the RNA-DNA hybrid

Predict the effect of a mutation that prevents sigma from dissociating from the RNA polymerase core.

its a lethal mutation

The DNA polymerase reaction

nucleophilic attack of the 3'OH to the inner most phosphate dNTP (the alpha phosphate of the nucleotide ie the closest to the ribose ring). creates a phosphodiester bond "attack on alpha-phosphate to create a phosphodiester bond"

Topoisomerase: where do they get their energy and the two types

online definition: A class of enzymes that alter the supercoiling of double-stranded DNA. (In supercoiling the DNA molecule coils up like a telephone cord, which shortens the molecule.) The topoisomerases act by transiently cutting one or both strands of the DNA. topoisomerase use ATP hydrolysis to relax DNA structures genetics definition: Type I topoisomerases (topol)- relax supercoils by breaking one strand, allowing it to rotate around the other strand & then rejoining the broken strand. "single strand break" - biochem Type II topoisomerases (gyrase) - cause a break in both strands. It can convert "positive" supercoils into "negative" supercoils. "double strand break"-biochem "topisomerases are localized near replication forks to take away the strain due to helicase activity due to polymerization reactions"

DNA polymerization direction? DNA editing direction?

polymerization is 5'-3' editing is 3'-5' Both DNA pol I and III have exonuclease activity to fix mistakes of nucleotide incorporation online: an enzyme which removes successive nucleotides from the end of a polynucleotide molecule.

types of RNA produced by RNA Pol II location?

produces mRNA, lncRNA, mrRNA find in nucleus

types of RNA produced by RNA Pol I location?

produces rRNA, find in nucleolus

types of RNA produced by RNA Pol III location?

produces tRNAs, snRNA and 5sRNA (produces the small RNAs) found in nucleus and sometimes cytoplasm

Amino acyl(AA)-tRNA synthetases:

readers of the genetic code because these enzymes add AAs to tRNAs. "recognize tRNA and responsible for putting correct AA on a tRN" online: Aminoacyl tRNA is a tRNA molecule that is bound to the A site of the ribosome, while peptidyl tRNA is a tRNA molecule that is bound to the P site of the ribosome. So, this is the key difference between aminoacyl tRNA and peptidyl tRNA.Nov

what is rate listing usually in transcription

recognition of promoter by the holoenzyme

histone deacetylases (HDACs).

remove acetyl groups

what do replicative DNA polymerases require? rate of dissociation? whats their role?

require" - a template strand - a primer with a free OH: (RNA is the in vivo primer, used for replication of both leading & lagging strands) - dATP, dGTP, dCTP, TTP & Mg2+ - are processive enzymes with slow rates of DNA dissociation - possess nuclease activity for removing mis-matched bases (proofreading)

An orthogonal genetic code

restricts the transfer of genetic information. Syn61D3 is resisted to viruses fished out of the River Cam in Cambridge. idk if theses important?

almost all eukaryotic RNAs are processed to be "matured" (post-translational maturation) the processing features depend on the type of RNA being processed what does each type pf RNA require?

ribosomal RNA (rRNA): RNase dependent "RNase is an enzyme that degrades RNA, counterintuitive that it requires this but it does" Base modification transfer RNA (tRNA): RNase dependent Base modification messenger RNA (mRNA): Capping "at 5' end of mRNA" snRNP dependent (splicing) Polyadenylation "set of adenines added at the end, another form of post-translational modification"

tRNAs

serve as adaptors between mRNA and amino acids during protein synthesis

the issue with single stranded DNA

single strand DNA can undergo intra-molecular hydrogen bonds and bind to itself (hairpins) single-stranded-binding DNA proteins (SBB) single strand DNA will form intramolecular H-bonds if not bound by single stranded DNA binding (SSB) proteins. single-stranded-binding DNA proteins (SBB) "protect single stand DNA by binding until polymerization can get to it. important to prevent intramolecular hydrogen bonding that would cause additional structures that helicase would struggle to break through"

which strand of DNA is read

the non-tempplate strand is the coding strand, it is the sense strand and thus the grownig strand

polyadenylation signal (poly-A)

"RNA is transcribing until it gets to the polyadenylation signal" "critical for making endonuclease cleavage and RNA pol II to dissociate"

fidelity of DNA polymerization (accuracy? of DNA polymeriziation)

"active site forms phosphodiester bond" - replication fidelity is enhanced ~1000 fold by proofreading/editing - DNA polymerases have 3"-5" exonuclease activity that removes mismatched base pairs. In E. coli, DNA Pol I proofreads - Editing is promoted by the instability of mis-matched nucleotide in the active site, causing migration of the base to the exonuclease active site - incorporation of an incorrect base-paired nucleotide stalls polymerase & increases the possibility of strand displacement to the exonuclease active site

when can elongation occur

"after synthesizing a little RNA, there is a loss of sigma" holoenzyme-->core enzyme, and core continues to cause elongation

why does RNAP bind inefficiently and transcription is at BASAL level after lac operon induction? what's our solution?

"because it has a bad promoter, so BASAL transcription level means LOW levels of transcription this happens becausee the promoter regions potentially not similar to the census region??" our solution: catabolite activator protein (CAP)

Chromatin remodeling complexes confer DNA accessibility

"promotor is buried in nucleosome" Transcription factor binds Transcription factor recruits co-activator Co-activator acetylates histone tails "HATs or mediators" Acetylation recruits chromatin remodeling complex Remodeling moves nucleosome to reveal promoter. GTFs bind and recruit RNA Pol II.

specificty of DNA incorporation in DNA replication

"shape is most important, then hydrogen bonding" - the structure of DNA polymerases resemble a right hand, with the palm serving as the active site - binding the correct dNTP (nucleotide) depends on H-bonding - dNTP binding induces conformational changes at the active site to form tight pocket that fits only correct base - shape complementarity of correct base pair is critical for phosphodiester bond formation, as analogs that share dNTP shape but not H-bonding can be incorporated

Splicing of pre-mRNA: recognition of splice junctions

*Depends on the U1 and U2 snRNPs: - U1 RNA base pairs with 5' junction. - U2 RNA base pairs with branchpoint sequence, which "activates" the branchpoint A.

maturation of mRNA: splicing Splicing of pre-mRNA: overview

- A spliceosome forms by association of all five snRNPs. - The snRNAs in the snRNPs play the central role in splice site selection and catalysis. - The snRNAs promote base pairing rearrangements that facilitate splicing. These base pairing changes are powered by ATP dependent helicases. - Two transesterification "formation of 2 regions of RNA" reactions occur: 1. to form an RNA lariat that has the 3' exon. (5' exon is released) "unusual phophodiester linkage" 2. tospliceexonstogetherandrelease the intron as a lariat RNA.

Attachment of an AA to a tRNA: activation of the AA what is it all catalyzed by? what does it form how many ATPs/energy needed

- AA activation is catalyzed by aminoacyl-tRNA synthetases: translators of the genetic code. - The AA is activated by forming aminoacyl adenylate (AMP). - Two ATPs are needed to form AA-tRNA. - Activation of the AA and transfer to a tRNA are catalyzed by the same aminoacyl-tRNA synthetase.

Attachment of an AA to a tRNA:

- AAs are added to the 3', CCA arm of tRNA using an ester linkage to the 2' or 3' OH of the ribose unit at the end of the tRNA. - The AA-tRNA or charged tRNA provides an activated form of the AA, as peptide bond formation is not energetically favored. - For each AA there is one activating enzyme (synthetase) and one or more tRNAs. Steps in AA-tRNA formation: "by synthetase activation" - activate the AA "make AA reactive by adding AMP" - transfer the activated AA to tRNA - proof-read the charged tRNA "synthetase is gonna-proofread the charging, if there is incorrect charging then the wrong codon AA"

Five major kinds of histones

- Are conserved, small, basic proteins. - Histone H1 is the linker histone. - H2A, H2B, H3, H4 form the nucleosome: - contain two domains: globular and tail. - associate as two H2A/H2B dimers and an H3/ H4 tetramer. "histones are somewhat basic meaning they are somewhat positive which helps with DNA compaction because DNA is negative"

Splicing of pre-mRNA: Remodeling of RNA interactions

- Association of U4-U5-U6 causes change - U5 holds two exons together.- U6 interacts with U2, releases inhibitory U4. - U6 also interacts with 5' splice junction, releases U1. -U2 bound at the branchpoint unpairs the branchpoint A for nucleophilic attack. - RNA rearrangements require ATP-powered helicases to unwind RNA duplexes and snRNP release.

DNA methylation

- Cytosine methylation correlates with transcriptional repression. - Cytosine methylation affects transcription by: - Interfering with binding of transcription factors - Platforms for recruitment of histone deacetylase enzymes. fund genetics: Methylation is histone post-translational modification on lysine or arginine Can also be done on lysine, the same wasy as the previous modification was done on lysine We add a methyl group, we han add 1-3 (can be added to each hydrogen & theres three hydrogens) We are changin the structure, thus attracting new proteins Methylation can be done in different levels on each amino acid; mono di or tri Methylation of lysine does not affect charge; but creates binding sites for other proteins that either activate or repress gene expression As a result od this modidifcaton, the protein types that bind are different Because the shape is different, the type of proteins that can bind or not bind is different in each case The protein bound to the methylated histone will vary on which histone residue is methylated & how much The affect of methylation vary. For example, methylation of h3 lysine residue 4 is associated with the activation, enriched near transcription start site. H3k9 or h3k27 methylations are associated with gene repression

topological stress cause by DNA unwinding what relieves this stress?

- DNA unwinding produces single stranded regions and the consequence of that is overwinding, this causes DNA strain (topological stress) topoisomerase relieve some of this stress

Acetylation

- Established by enzymes called histone acetyl transferases (HATs) - Neutralizes the positive charge on lysine. - Found on genes with active transcription. - Prevents interactions between histone tails and DNA and between adjacent nucleosomes. - Serves as a platform for recruitment of general transcription factors (GTFs), such as a TAF1, a bromodomain TF subunit of TFIID or chromatin remodeling complexes. - Reversed by histone deacetylases (HDACs). fund genetics : Acetylation—above is a lysine with an NH3 with a pisitive charge, acetylation adds an acetyl group which neutralizes the charge (+--> -), this has an affect on the histones as DNA is charged negatively as it contains a phosphate backbone, non-modified lysine causes electrostatic interactions between the + on the histones & the - DNA, making everything stick together better. When modified, theres less charge on the hsitone & thus less hold on the nucleosome which leds to activation of gene as it releases the tails from interacting with the DNA here

- Trans-acting factors

- General transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH - Mediator - Histone modifying/ remodeling complexes

regulation of the lac operon when is it induced

- Lactose is transported into the cell. - Produces inducer (allolactose). - Inducer binds and inactivates Lac Repressor. - Inducer changes the structure of the Lac Repressor protein reducing its ability to bind to DNA. - RNAP binds inefficiently. Transcription at a BASAL level. "get rid of lac repressor via lactose presence. because lactose forms an inducer, binds to the lac repressor and inactivates it"

Pol II regulation: building a pre-initiation complex steps summary

- TFIID begins assembly (TBP, TAFs) by promoter identification. - TFIIA stabilizes TFIID, bending DNA. - TFIIB recruits Pol II bound to TFIIF. - TFIIE binds, recruits TFIIH - TFIIH unwinds the promoter (helicase) and phosphorylates Pol II CTD (kinase) at Ser5 "RNAP II is a small part of a. while set of GTFs that are helping recruit and position RNAP II" "when TFIIH is recruited, it has two helpful properties for transcription, it 1) contains a subunit that's a helicase (uses ATP-energy), and that's gonna help make the transcription bubble, help the open complex, the open complex to help identify the template strand to start RNA synthesis. the 2) is it contains a kinase that phosphorylates the CTD ((carboxyl terminal domain) to begin to allow Pol II to get its properties it needs to start elongation. so, go from open to closed, o the open the complex has NTPs? (nucleotides?)that come in initiationlyy transcribing and then you have an elongation-complex"

telomerase and disease

- Telomeres are proposed to form a structure resulting from single stranded invasion of duplex DNA to form a loop, which is stabilized by telomere binding proteins .-Mutations in two telomerase core components cause disease, 1) telomerase RNA and 2) telomerase reverse transcriptase (TERT). In these diseases, telomeres become very short in highly prolific cells, germline and stem cells. - Telomerase is low or absent in normal cells. Cancer cellsinduce telomerase expression .- Telomere shortening is associated with aging.

transcription and mRNA maturation are coupled

- The CTD on the largest RNAPII subunit coordinates transcription and processing. - Most splicing occurs co- transcriptionally. - CTD phosphorylation associated with co-factor recruitment: - Ser 5: Capping machinery - Ser 2: Poly A machinery - Ser 2/5: Splicing

Regulation of the lac operon: Why is CAP needed?

- The lac promoter deviates from the consensus, lacks UP element. - s70 recognition is lowered, need help for RNAP recruitment. - Low glucose causes high cAMP, CAP binds DNA - CAP interacts with RNAP, improves promoter recognition. - Loss of CAP binding in presence of glucose (low cAMP) called Catabolite Repression.

Splicing of pre-mRNA: Spliceosome formation

- The spliceosome forms when the U4-U5-U6 complex joins the mRNA. - Formation is coupled with RNA and protein reorganization. - Reorganization: starts when U6 disengages from U4 and base pairs with the 5' junction, releasing U1. Next, U6 base pairs with U2 to bring the 5' splice site close to the branchpoint. - The U2 and U6 snRNAs form the catalytic center of the spliceosome. - Exons are held together by U5.

wobble hypothesis

- There is at least one tRNA per AA, but fewer than one tRNA per codon (i.e. fewer than 61). - Some tRNAs recognize more than one codon, known as wobble: - Features of wobble, the codon-anticodon interaction: 1) The last two bases of an anti-codon base pair in a standard way. Recognition is precise. 2) The first base of an anti-codon determines whether the tRNA will read one, two or three codons: - C or A: one codon, U or G: two codons, I : three codon "remember the last two of the anticodon as the first two of the codon" The wobble position of a codon refers to the 3rd nucleotide in a codon. This nucleotide has two major characteristics: Binding of a codon in an mRNA the cognate tRNA is much "looser" in the third position of the codon. This permits several types of non-Watson-Crick base pairing to occur at the third codon position.Jan 2, 2010

trp operon

- Trp operon encodes biosynthetic enzymes for making tryptophan. - mRNA is produced when tryptophan levels are low.

Characteristics of the genetic code what kind of overlap? what is it comprised off? which direction is it read? importance of mRNA cap?

- is comprised of triplets. - is non-overlapping and read sequentially - contains no punctuation between codons. - has direction: codons are read 5' to 3'. "mRNAs capping is important for translation initiation because cap is what is first identified by the initiation factor for tranlsation" - is degenerate: more than one codon encodes the same AA. When several codons are used, the difference is usually in the third base of the codon: ALA: GCU, GCC, GCA, GCG - is nearly universal.

DNA polymerase III

- is the major DNA polymerase for synthesizing DNA - consists of 2 copies of each other core enzyme (the polymerase subunit), the exonuclease subunit, the clamp loader, the processive factor (sliding clamp, B2) plus additional subunits) - catalyzes 1000 nucleotides per second "dimer helps coordinate synthesis of both strands, had 3'-5' exonuclease activity"

DNA replication general overview

- is the process of copying one DNA into two - involved proteins that have seven different enzymatic activities - requires proteins that identify where to start, or the origin of replication

how does RNA synthesis occur

- occurs in the 5'-3- direction "template is read 3'-5' while the RNA is made from 5'-3'" - requires a DNA template, NTPs, divalent cation "notice it uses NTPs instead of dNTPs" - does not require a primer "major difference between DNA replication and RNA transcription" - starts at +1

Nucleosome structure

- octamer of histones - DNA forms a left-handed helix as it wraps 2x around the nucleosome - individual histone tails extend away from the nucleosome -transcriptoinal regulation involves post-translational modifications of AA in the tails "tails are highly modified, stat to open ip and read the DNA better"

rRNA (ribosomal RNA) maturation

- rRNA synthesized as a single precursor RNA. "single transcript" - Pre-RNA (45S) processing involves: - Methylation predominantly at ribose 2' OHs in mature RNA that is directed by snoRNPs (small nucleolar RNA proteins associated with one small RNA). "direct methylation of the sequences we want to have" - Assembly into ribonucleoprotein complex with ribosomal proteins - Cleavage by RNase III to produce smaller rRNAs.

replication of liner chromosomes

-Removal of the primer at the 5' end of the lagging strand leaves a single strand DNA end. -Replication of the shortened lagging strand leads to lossof DNA (~10 to 50 nucleotides each cell cycle). -Cells have evolved a mechanism to ensure that importantDNA sequences are not lost: Telomerase .- Telomerase adds hundreds of G-rich repeats onto the end of chromosomes. After every round of DNA replication, the chromosomes will get a little shorter "information lost at the end, ~50 nucleotides at the end of the cycle so the solution is tolmerase building telomeres at the end of chromosomes, adds a bunch of G-protein rich repeats at the end and this allows an extension to protect the vital information" fundamental genetics: Telomerase carries a short RNA molecule that acts as a template for the addition of DNA at the 3' end This is an example of reverse transcription (RNA-->DNA)

Pol II regulation: TFIID and promoter recognition

-TFIID - Is a general transcription factor that recognizes RNA Pol II promoters. - Contains TBP (TATA binding protein) that binds the TATA box. - Contains TBP-associated factors (TAFs) that bind the Inr and DPE in TATA-less promoters. - Primes the promoter for assembly of RNA Pol II GTFs. "TFIID is basically the sigma factor for RNAP2 in prokaryotes"

The platform for reading the code: Ribosomes

-The E. coli ribosome has two subunits: - 50S subunit- 34 proteins (L1-L34, red) and 23S (yellow), 5S rRNAorang - 30S subunit- 21 proteins (S1-S21, blue) and 16S rRNA (green) - RNA makes up 66% of mass of ribosome and constitutes the catalytic portion of the ribosome. - Ribosomes are ribozymes - Ribosomes make up ~40% of mass of E. coli cell.

small differences between genetic codes

1. Ciliated protozoans use only one stop codon. 2. Mitochondria have differences in the code - Has 4 stop codons - Permitted since mitochondria encode own set of tRNAs. "mitochondria has its own genome which encodes for its own tRNA"

three stages of transcription and what happens in each stage

1. Initiation: - Promoter recognition by the holoenzyme (often rate limiting) - Transition from closed to open complex 2. Elongation: - Loss of sigma (s) - Core enzyme continues 3. Termination: - Factor independent by DNA structure - Factor dependent by Rho

2 The critical elements of the genetic code

1. tRNAs:readers of the code because these RNAs are the link between the mRNA and amino acids (AAs). 2. Amino acyl(AA)-tRNA synthetases: readers of the genetic code because these enzymes add AAs to tRNAs.

replication fork

A Y-shaped region on a replicating DNA molecule where new strands are growing. - the sire of DNA synthesis is called the replication fork - DNA synthesis of both strands occur at the same time - the replication fork moves in 5" to 3" direction " DNA pol I--> removal of primer from the fragments and replacing the gaps by relevant nucleotides (proofreading/primer removal) with exonuclease activity from 3'-5', 5'-3'" "DNA pol III --> mainly involved with the synthesis of the leading and lagging strands. only 3'-5' exonuclease activity, aids in base pairing of incoming nucleotides with the template strand. if any mutation is in the template, will be removed and resynthesized."

cis acting elements of promotors

Cis-acting elements: - Promoter: TATA, Initiator (Inr), Downstream Promoter element (DPE) - Proximal promoter elements: CAAT box, GC box - Enhancers, Silencers: long distance acting "can act on 1k-2k nts away"

example Synthetase attachment of THR to a tRNA: getting the AA right

Cooperation between synthetase sites: - active site: - Zn+2 contacts THR hydroxyl and amino groups, an ASP contacts hydroxyl. Valine lacks hydroxyl, so will not bind. "rejects the larger AAs" - editing site: - Thr-tRNAThr will not fit due to presence of methyl group, but Ser-tRNAThr will fit. "allows the smaller AA initially but when there is transfer to the tRNA binding site the smaller AA are eliminated" Actions of active and editing sites serve as a double sieve: - active site rejects larger AA than the correct one. - editing site accepts smaller AAs than the correct one.

the bacterial replication of origin

DNA replications begins at an origin of replication - DnaA identifies the origin, binds four sites in the elee=ment, and olgiomerizes, wrapping origin DNA using energy of ATP - DnaB (helicase) unwinds DNA using energy of ATP. - single strand binding (SSB) proteins join and prevents reannealing at the origin to form the prepriming complex (all above steps happen before primer is present) - primase synthesizes RNA to establish primer

central dogma

DNa-DNA is replication DNA to RNA is translation RNA to protein is transcription

3. Termination

Events in intrinsic transcription termination: - The DNA signal is a GC-rich palindrome followed by >4 Ts. - RNAP pauses once transcribes a string of Us - RNA upstream of Us forms a hairpin or stem-loop structure - Hairpin plus the unstable U-A RNA-DNA hybrid allow RNA dissociation from template. "when RNA pol hits a string of Ts and starts to incorporate Us string, it slows down and pauses so then the upstream sequence has time to form this step-loop. as a consequence, the amount of RNAthats bound to the template is reduced and its a weak DNA/RNA hybrid. bc of weak hybrid, that RNA is released. so hairpin + unstable U-A-RNA/DNA hybrid= mRNA released, pol is then terminated"

what causes Okazaki fragments

Generally, DNA polymerase adds nucleotides in the 5' to 3' direction. Since the leading strand runs in the 3' to 5' direction, the enzyme can continuously add nucleotides to the growing strand on the leading strand. However, since the lagging strand runs in the 5' to 3' direction, the chain growth of the newly-synthesizing DNA strand is paused when it reaches the 5' end of the strand. Then the synthesis of another DNA strand begins at the replication fork. The replication fork is the position on the DNA double-strand where the unwinding begins. Unwinding is critical in the synthesis of new DNA strands on the original strands. Once the replication fork moves forward on the DNA double-strand, DNA polymerase can add nucleotides onto the lagging strand. However, the synthesis is paused when it reaches 5' end of the RNA primer of the already-synthesized DNA stretch. Hence, the DNA synthesis at the lagging strand is discontinuous and the resultant DNA stretches are known as Okazaki fragments. Conclusion Okazaki fragments are the short DNA fragments on the lagging strand formed during DNA replication. Since the lagging strands run in the 3' to 5' direction, the DNA synthesis on the lagging strand is discontinuous. It forms Okazaki fragments on the lagging strand that are ligated later by DNA ligase. ONLINE DEF

mRNA (messenger RNA) maturation

Includes 5' capping Splicing (introns removed) 3' polyadenylation "As added" RNA editing "without caps added or adenylation, the mRNA wouldn't be viable and would be removed"

Pol II regulation: transcription factors recruit: Mediator

Mediator is: - an intermediary between transcription factors and RNAPII - recruited by transcription factors (activators, repressors) to influence the activity of RNA Pol II. - promotes phosphorylation of Ser5 to promote RNA Pol II initiation. is is a type of coactivator

The genetic code

The genetic code is the code that correlates a sequence of three nucleotide bases (codon) with a specific amino acid. "start=AUG=methionine" each is read vis an anticodon there is a peptide bond formation

why we have diff types of cells

Transcriptional regulation underlies differentiation - Differentiation involves large scale genome silencing, permitting cell type specific patterns of gene expression. - Somatic cells (fibroblasts) can be re-programmed toward pluripotency by inducing expression of four key transcription factors

Splicing of pre-mRNA: transesterification

Transesterification: reaction between alcohol and ester to form a different alcohol and ester. First transesterification: formation of unusual 5'-2' phosphodiester linkage, called the lariat. Second transesterification: formation of 5' to 3' phosphodiester bond that links exons- the spliced product.

RNA Pol II function

makes snRNA (splicing), snoRNA( modifies rRNA), miRNA, siRNA (help stop protein production), piRNA, lincRNA and mRNA!!!!!!!!

splicing by fund genetics

splicing- RNA processing that removes the introns Much greater diversity of proteins can be achieved by alternative splicing (70% of all guman genes are being alternatively spliced) More complex organisms also have more introns per gene In humans, that leads to production of more than 70,000 types of proteins out of 21,000 genes More complex=more alternative splicing Lots of regulatory rna that play very important roles Splicing involves recognition of specific sequences on the RNA & snRNPs All introns require GU at the 5' & AG at the 3' & A in between (closer to 3'). Larger sequence motifs are conserved within species SnRNPs= small nuclear ribonucleo protein complexes. The snRNPs involved in spicing are Us (U1, U2, U3...) SNRNPS & Usz: the RNA is the catalytic converters, proteins are supporting functions. Uses base pairing RNA U to identify different regions during the splicing process. Us in the 5; splice with the A branch point. Next step, more Us, more snrps that dissociate with RNA & form a splicosome which is the complex that catlayizes splicing, then the first step Formation of lariat after the attack site, the lariat site causes a covalent bond between the first site & the intron, creating a three way branch. Once this occurs we have a free OH that attacks the 3' splice site, which results in the police product. The spliced sites will be rejoined & the lariat will be trashed as we no longer need it. The spliced exon will now go to the rna that will be transported & translated ??