Molecular Cell Biology Exam 2 Review

The processing of the three largest rRNAs (the 28S, 18S, and 5.8S)"

"involves the initial synthesis of a long precursor transcript, the cleavage of the precursor into smaller products, and the subsequent modification of specific residues in the resulting products"

Levels of transcription-basal

(+); Stimulated by Cis-acting sequences bound by Activators: up to (+++); Stimulated by Enhancers bound by Activators: (+++++)

Role of the promoter -what does it do? Why is it essential?

-DNA sequence to which RNA-Pol binds to initiate transcription of a gene -Directs RNA Polymerase to the template -Dictates which of the two DNA strands will serve as the template -Positions RNA Polymerase over the initiation site Serves as "ON" switch for transcription

What are subsequent events of translation?

-Shortening of the poly(A) tail (every time the mRNA is translated, the tail gets shorter). -Degradation by the exosome (when the poly(A) tail becomes too short the exosome degrades mRNAs from the 3' end), as well as by 5' decapping followed by a 5'-3' exonuclease activity.

elongation (transcription)

-The covalent addition of nucleotides to the 3' end of the growing polynucleotide chain; this involves the development of a short stretch of DNA that is transiently single-stranded

open reading frame (ORF)

-Three nucleotides encode from one amino acid -(4)^3 = 64 different potential combinations for only 20 amino acids -Redundant Degenerate -Extreme care must be placed on selecting the right ORFs otherwise may end up with something that is completely unreadable or a "nonsense."

role of sigma subunit in prokaryotes and eukaryote equivilant

-To direct polymerase to promoters by binding specifically to both -35 and -10 sequences leading to initiation of transcription. -Eukaryotes- transcription factors for RNA Pol II to initiate transcription

Are there any differences between eukaryotic and prokaryotic ribosomes?

-Yes compositions are different -Eukaryotic ribosome - 80S: small subunit 40S, large subunit 60S -20S, 5.8S, and 5S present in the 60S -18S present in the 40S subunit -Prokaryotic ribosome - 70S: small subunit 30S, large subunit 50S -16S only subunit in prokaryotes

Termination

-different for every RNA-Pol; for RNA-PolII termination occurs after PolyAdenylation and is triggered by it

why are mutations important? do they play a role in evolution?

-most are deleterious , and can lead to hereditary diseases or cancer. -Yes, mutations drive evolution

what is the difference between transcription-coupled nucleotide excision repair and general genomic nucleotide excision repair

-transcription coupled repair involves recognition of stalled RNA polymerase by CSB & CSA which is not in general excision repair. -Transcription coupled repairs active DNA -general repairs silent DNA

Pay special attention to the following methods of translational regulation:

1) Translational repressors (proteins that associate with transcripts and repress translation) 2) miRNAs 3) Phosphorylation of eIF2 and eIF2B 4) Phosphorylation of 4E-BPs

Following nuclear export, mRNA is Translated (in association with Ribosomes), and then it decays - DEGRADATION. -What are the mechanisms of mRNA Degradation?

1. Associated to errors in splicing - Non-sense mediated mRNA decay (NMD). 2. Cytoplasmic - a. Decapping pathway. b. Deadenylation-dependent pathway (Exosome). c. Endonucleolytic pathway.

What does mRNA processing involve?

1. Capping. 2. Start splicing. 3. Polyadenylation (addition of ~200 A at the 3' end). 4. Transcriptional termination (brought about by Polyadenylation). 5. Finish Splicing. 6. Nuclear export.

What are the two stages at which translational fidelity is determined & ensured?

1. Charging of tRNA by aminoacyl tRNA synthesis 2. Pairing of anticodon in tRNA with the codon in mRNA

What does tRNA processing involve?

1. Cleavage (@5' and 3' - the 5' cleavage is executed by a RIBOZYME [RNase P]) - What's a Ribozyme? 2. Addition of a CCA sequence at the 3' end. 3. May involve splicing (some tRNAs are spliced!). 4. Base modification. 5. Loading of an amino acid at the 3' end by an Amino-Acyl Synthetase.

Splicing mechanism

1. Cleavage at 5' splice site (GUAG) , formation of lariat like intermediate 2. Cleavage at 3' splice site; ligation of exons 3. Transesterification reaction 1 (5'-2') 4. Transesterification reaction 2 (5'-3') 5. spliced mRNA

Activators are MODULAR proteins (they contain 2 DOMAINS) - What are those two domains? Why is modularity important?

1. DNA Binding Domain-interact with mediator and general transcription factors to facilitate the assembly of the transcription complex and stimulate transcription 2. Activation Domain-interact with coactivators that facilitate transcription by modifying the chromatin structure

what are the two main types of spontaneous mutation? why are they important?

1. Deamination and 2. Depurination deamination happens frequently with B and T cells and can turn a cytosine into a uracil depurination causes a purine group (A or G) to remove from the sugar alters normal pattern of complementary base pairing

What are the players involved in translation?

1. Messenger RNA (mRNA) 2. Transfer RNA (tRNA) & Aminoacyl-tRNA synthetases 3. Ribosomal RNA & Ribosomes 4. Protein Factors (Initiation, Elongation, & Termination Factors)

what are two examples of direct reversal of DNA damage? which one is present in humans

1. Photo reactivation 2. Repair of O^6 methylguanine methyltransferase (in humans)

Sequence Elements that regulate binding/initiation: -DNA Sequences involved // Protein factors that recognize each one of them:

1. Promoter // General Transcription Factors. 2. Cis-acting sequences (proximal control elements) // Specific Transcription Factors or ACTIVATORS. 3. Enhancers // Specific Transcription Factors or ACTIVATORS. 4. Silencers // Specific Transcription Factors that act as REPRESSORS.

How is the start codon selected in eukaryotic cells? What's the Kozak Sequence?

1. Ribosomes recognize most eukaryotic mRNAs by binding to the 7-methylguanosine cap at their 5' terminus and then scan downstream of the 5' cap until they encounter the initiation codon (usually AUG) 2. Kozak sequence -Kozak consensus sequence plays a major role in the initiation of the translation process -a sequence which occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG - (A/G) AUGG translation (A/G: -3) (AUG: +1) (G: +4)

Regulation is exerted at five different levels:

1. Sequence Elements that regulate binding/initiation 2. Regulating DNA condensation (packaging) 3. Elongation (mediated by P-TEFb recruitment) 4. Regulating Processing (of the transcript) 5. Regulating transcript stability

Binding stage: Factors involved and what they do

1. TFIID(TATA-Binding Protein[TBP]+TATA-Associated Proteins [TAPs]) -TBP induces a bend in the DNA that creates a clear mark in the area containing a promoter 2. TFIIB (B=bridges interaction with RNA-Pol II) 3. RNA-Pol II+TFIIF 4. TFIIE+TFIIH (TFIIH contains 2 different activities, HELICASE [exerted by the Xeroderma pigmentosum proteins XPB and XPD] and KINASE activity and is required to unwind the DNA to start transcription and to phosphorylate the Carboxyl-Terminal Domain or CTD in RNA-PolII) 5. Phosphorylation of the CTD of RNA-PolII releases the RNA-PolII from the pre-initiation complex and allows active transcription to start

How is transcription termination achieved in prokaryotes

1. Termination Sequence - symmetrical inverted repeat of a GC-rich sequence followed by four or more A residues Results in the formation of a stable stem-loop structure. 2. Rho - ATP-dependent unwinding enzyme (moves along the newly formed RNA molecule towards its 3' end and unwinds it from the DNA template). Binds extended segments (>60 nucleotides) of single strand DNA

What's the chemical group used to link the amino acid residue to the tRNA? Why is that relevant?

1. The Carboxyl group; 2. It determines the direction of protein synthesis, from Amino (which is free for the first residue incorporated) to Carboxyl end

Differences between eukaryotic mRNA and prokaryotic mRNA

1. prokaryotic do NOT have a Cap o Shine-Delgarno sequence o Located at very short distance from the start codon o Polycistronic- code for more than one protein (multiple protein) 2. Eukaryotic need a Cap o 5'end can be far away from the start codon o Kozak consensus sequence charge allows the recognition o Monocistronic -encodes for a single protein (regulating every single component) allow more flexibility

which rRNAs are encoded in the same transcriptional unit?

18S, 5.8S, and 28S

how many different types of antibodies are produced by each B cell?

24 million 24,000 by heavy chains 1,000 for light chains

How is the mRNA read in translation?

5' to 3' direction

Which rRNA is encoded in a separate gene in a different chromosomal location?

5S rRNA

What is capping? When does it occur?

A 5′-cap is added to the 5′-end of newly synthesized mRNA using the modifield nucleotide, 7-methylguanosine. Capping occurs shortly after initiation of synthesis of the mRNA and precedes other modifications that protect the mRNA from degradation by RNases.

What's a codon?

A codon is a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis. (One of 64 triplets)

Promoter

A specific nucleotide sequence in DNA that binds RNA polymerase and indicates where to start transcription

How is DNA condensation regulated?

A. Post-translational modifications of HISTONES B. Nucleosome Remodeling Factors C. miRNAs D. Chemical modifications of DNA - DNA METHYLATION

What's wobble base pairing (also known as the Wobble mechanism)? Why is it important?

A1/ The ability to establish non-standard or non-Watson-Crick base pairings at the third position of the codon; A2/ Its relevance is to allow a single tRNA to recognize different codons

-Besides keeping the small ribosomal subunit away from the large, what else does eIF3 do?

Allows the interaction with the mRNA - specifically with eIF4G

__________________provides an important mechanism for tissue-specific and developmental regulation of gene expression

Alternative splicing

Why is that important from the human health point of view?

Antibiotics: inhibit a key function in bacteria without affecting cellular function in humans • Penicillin- cell wall synthesis • Ribosome- targeted antibiotics

Where are silencers located?

Are typically located far away from the promoter (100,000 bp away) Can be located upstream or downstream from the core promoter

How is GTP-loaded (or bound) eEF1alpha recycled? What's the name of the GTP-Exchange Factor for this process?

As elongation continues, the eEF1α that is released from the ribosome bound to GDP must be reconverted to its GTP form. The GTP-Exchange Factor is eEF1βγ, which binds to the eEF1α/GDP complex and promotes the exchange of bound GDP for GTP, resulting in the regeneration of eEF1α/GTP that is now ready to escort a new aminoacyl tRNA to the A site of the ribosome - beginning a new cycle of elongation.

Deamination of cytosines produce a type of DNA damage that is most frequently repaired via

Base excision repair

Why does scanning require ATP hydrolysis?

Because it requires energy to do so

How is the activity of Activators regulated?

By regulating their cellular levels, cellular localization, & post-translational modification.

This is the correct order of the events that mRNAs undergo during their life cycle

Capping, splicing, polyAdenylation, finish transcription, finish splicing, nuclear export, translation, decay

8. DNA-Methyl transferases - what do they do? what's their effect in DNA packaging?

Controls transcription; DNA is methylated specifically at the cytosines that precede guanines in the DNA chain (CpG dinucleotides), and this methylation is correlated with transcriptional repression; associated with transcriptional repression of some genes, in concert with alterations in chromatin structure

template for transcription

DNA

How is the enormous number of antibody molecules produced by our body generated at the genetic level?

DNA rearrangements and site specific recombination

DNA mutations occur a. as a result of environmental factors b. at random C. from sun exposure D. over time E. All of the above

E. all of the above

RNA polymerase

Enzyme that synthesizes RNA from a DNA template during transcription.

How do lncRNAs contribute to the regulation of DNA packaging? What is the name of the enzyme that is recruited by lncRNAs? What does it do?

Enzyme= DNA methyltransferases are a group of enzymes involved in the regulation of gene expression. (repression) lncRNA direct repressive chromatin modifications to specific regions in the genomes

In eukaryotic cells, transcription of the genes coding for the larger ribosomal RNAs (rRNSs) yields a large 45S pre-rRNA that is later on processed to produce the 18S, 5.8S, and 5S rRNAs"

False

Sp1 is an example of a transcriptional repressor

False

There are fewer enhancers than genes in the human genome

False

Where are enhancers located?

Far away: sometimes 100s of kilo base pairs from the transcription start site: up or downstream from a gene

Before coming to class you spent 15 minutes laying down under the beautiful El Paso sun. During that time, the most likely type of DNA damage suffered by your epithelial cells is

Formation of thymine dimers

how many gene segments are used to code for the heavy chain? how many different types are there for each of these segments?

Four gene segment types V (variable): 500 segments D (diversity ): 12 segments J (joining): 4 segments C (constant): 1 segment =517 segments

Transcription factors: General vs. Specific

General - involved in transcription from all promoters (basic transcription machinery) Specific - bind to DNA sequences that control expression of individual genes (regulation of transcription)

can homologous recombination be considered to be a DNA repair mechanism? why?

General homologous =yes repairs double stranded breaks in meiosis sits specific= no used to generate immunity cells

histone methylation that always stimulates

H3K4me3

histone methylation that always inhibits

H3K9me3

Histone acetylation is mediated by

HAT

Histone ACETYLATION & DE-ACETYLATION - Histone Acetyl-Transferases (HATs) & Histone De-Acetylases (HDACs) - How do they affect DNA packing? Why?

HATs are characteristic of actively transcribed chromatin (alters CHARGE of histones thus affecting their ability to form a tight closed structure with DNA) whereas HDACs are associated with inactive chromatin that cannot be transcribed; transcriptional activators and repressors are associated with coactivators and corepressors, which have HAT and HDAC activities, respectively. Hats- remove the positive charge from lysine which relaxes chromatin.

What's the name of the different regions in the ribosome that are involved in tRNA binding?

Has 3 sites = P (peptidyl), A (aminoacyl), and E (exit) sites

Why is rRNA important?

Heart of the ribosome; RNA; recognition of correct codon-anticodon pairing in the decoding center, like peptidul transferase activity, is principally based on rRNA activity

Histone Methyl-Transferases (H-MTs) - How do they affect DNA packing? Why

Histone methylation is in general associated with transcriptional repression. However, methylation of some lysine and arginine residues of histones results in transcriptional activation. Does NOT affect charge - Instead, it provides a binding surface for various activators or repressors; Methylation Histones can lead to both increased transcription or decreased transcription

What's a Reading Frame?

In molecular biology, a reading frame is a way of dividing the sequence of nucleotides in a nucleic acid (DNA or RNA) molecule into a set of consecutive, non-overlapping triplets. Where these triplets equate to amino acids or stop signals during translation, they are called codons.

How can miRNAs affect DNA packing? What's their ultimate effect? What's the name of the enzyme that mediates that effect (the effect on DNA packaging)?

Induces histone modifications that lead to chromatin condensation and the formation of heterochromatin, repressing transcription; enzyme=histone methyltransferase

What's involved in creating a new peptide bond?

Insertion of a correct aminoacyl tRNA into the A site triggers a conformational change that induces the hydrolysis of GTP bound to eEF1α and release of the elongation factor bound to GDP. Once eEF1α has left the ribosome, a peptide bond can be formed between initiator methionyl tRNA at the P site and the second aminoacyl tRNA at the A site. This reaction is catalyzed by the large ribosomal subunit, with the rRNA playing a critical role.

met-tRNA - why is always Met the first amino acid used to start translation in eukaryotic cells?

It is the only one with the ability to bind to the P site directly

Role of P-TEFb?

It neutralizes the negative action of NELF and DSIF by binding to RNA Pol II, releasing NELF and phosphorylating DSIF that further neutralizes its inhibitory activity on RNA Pol II. It's positive action drives RNA Pol II into the highly processive elongation stage of RNA transcription.

The factor that links the general transcription factors with the specific transcription factors is

Mediator

Many transcripts are "frozen" at the early stage of transcription (about 50 nclt away from the +1 position) -The halting of RNA-Pol II is mediated by a set of 2 factors. What are their names?

NELF & DSIF

Role of DSIF and NELF? ? Why is it important to halt the RNA-Pol II within about 50 nucleotides of the transcriptional start site? What is achieved with that?

NELF and DSIF produce a dramatic slow down in RNA POL II in transcription, resulting in a complete pause right around the time when capping takes place. Capping is a process that takes place early during transcription and essential to ensure survival which would otherwise be degraded by exonucleases

Are primers required for transcription?

No

Silencers - are they similar to activators? How can they inhibit transcription? Can they affect the activity of activators?

No, silencers Inhibit transcription (negative regulation), which is the opposite of activators. Silencers recruit histone de-acetylases (HDACs) in order to inhibit transcription; yes they can affect activators

Template for transcription in eukaryotes is organized in NUCLEOSOMES - why is that important?

Nucleosomes serve as general gene repressors. They help assure the inactivity of all genes in eukaryotes except those whose transcription is brought about by specific positive regulatory mechanisms. condensed or decondensed chromatin

The way that your body most likely dealt with the damage indicated in the last question is

Nucleotide excision repair

transcriptional regulation

Occurs several different stages during the process of transcription, but a substantial amount of regulation occurs at the Binding/Initiation stage.

What is the factor that phosphorylates NELF & DSIF? What is the role of that factor?

P-TEFb- contains protein kinases that phosphorylates NELF & DSIF as well as serine-2 of RNA polymerase CTD. This leads to productive elongation

What is the evidence that DNA packing (condensation) is related to Transcriptional Regulation (degree of condensation dictates transcriptability)?

Packaging of eukaryotic DNA into chromatin limits its availability as a template for transcription, so chromatin structure is a critical aspect of gene expression in eukaryotic cells. Rather than being present within the nucleus as naked DNA, the DNA of all eukaryotic cells is tightly bound to histones. The basic structural unit of chromatin is the nucleosome. The chromatin is further condensed by being coiled into higher-order structures organized into large loops of DNA.

It is the year 2018 and the Chinese government has embarked itself in an ambitious program to improve the human species by "adding" things that are lacking in our genome thru CRISPER/Cas9. If you could guess (based on current MCB knowledge) what changes might have been implemented, the Chinese government is very likely to include _____ as one of their first targets for insertion into the human genome

Photolyase enzyme activated by UV radiation to initiate direct reversal repair thru photoreactivation humans and placental animals lack photolyase

Polysomes vs. Monosomes - why does it matter? What's a polysome?

Polysomes consist of mRNAs occupied by two or more ribosomes, whereas monosomes are a mix of mRNAs bound by a single ribosome plus ''vacant couples'' A polysome is also known as a polyribosome - it is the group of ribosomes bound to an mRNA molecule that translate it simultaneously; spaced at intervals of about 100-200 nucleotides. mRNAs are usually transcribed in this manner, where each ribosome within the group functions independently to synthesize a separate polypeptide chain.

what is the role of Rad51

Promote strand invasion with the single stranded DNA overhang generated during the first event (exonuclease digestion to generate 3' overhang) eukaryotes

once the RNA-PolII has disengaged from the pre-initiation complex (which contains XPB and XPD), how does RNA-PolII manage to open the dsDNA to continue reading the template?

RNA Pol II is associated with mediator proteins, as well as with general transcription factors at the promoter. The mediator complex binds to the nonphosphorylated CTD of POL II and is released following phosphorylation of the CTD when transcription initiates. The phosphorylated CTD binds other proteins (elongation and processing factors) that facilitate transcriptional elongation and function in mRNA processing.

If you wanted to specifically inhibit the translation of one specific mRNA, what would be the best approach?

RNA interference.

What are the enzymes involved in the process bound to? (In other words, what's the structure that recruits the capping enzymes to the mRNA?).

RNA pol II recruits capping enzymes to mRNA

what is the enzyme involved in transcription?

RNA polymerase

"In eukaryotic cells, the RNA-Polymerase that transcribes the 5S ribosomal RNA (rRNA) is

RNA-Pol III

Types of RNA-Polymerases & specific functions.

Remember, all you need is to know that RNA-Pol I transcribes Ribosomal RNA [rRNA-all except 5S which is synthesized by RNA-Pol III], RNA-Pol II transcribes messenger RNA [mRNA], and RNA-Pol III transcribes transfer RNA [tRNA] and the 5S rRNA.

what stage of the cell cycle is it most likely to occur?

S & G2 phases (sister chromatids visible)

IF2-GTP - role in the process?

Sensor; bring mRNA will also bring the tRNA

Who recognizes the promoter first in eukaryotic transcription

TBP, which is part of TFIID

RNA pol II & TFIIF

TFIIF helps RNA pol II associate to other factors already placed on DNA

The helicase activity that allows transcription by RNA-Pol II to start is mediated by

TFIIH

It would be more damaging for the cell to undergo mutations that disrupt the 3'-5' Exo activity of DNA POl than to undergo mutations that disrupts enzymes involved in DNA repair mechanisms

TRUE

What's the role of the 5' cap and the 3' poly(A) tail on translational initiation?

The 5' cap and 3' poly(A) tail ensure mRNA stability; 5' cap is required for translation whereas 3' poly(A) tail only enhances translation and is not necessarily needed

If you wanted to inhibit translation (all together, for all possible transcripts) which would be the best alternatives?

The best alternatives would be phosphorylating eIF2 or eIF2B by regulatory protein kinases or by eIF4E binding proteins (4E-BPs), which bind to eIF4E and block its interaction with eIF4G.

Where are core promoters located?

The core promoter region is located most proximally and contains the RNA polymerase binding site, TATA box, and transcription start site (TSS).

Transesterification - What is it? When does it happen?

The formation of a new phosphodiester bond between nucleotides that were previously bound to other nucleotides; occurs in the joining of exons

Why is the genetic code said to be DEGENERATE?

The genetic code is degenerate because more than one codon sequence can code for the same amino acid. ... Codons that specify for the same amino acid are called synonyms.

How is transcription by RNA-PolII different from that driven by other RNA-Pols?

The location of the core promoter varies and the general transcription factors involved are different; the processing of the transcripts generated is also different and the process by which termination is achieved is also different.

Mechanisms by which translational regulation can be achieved - Which ones are global and which ones are specific to one transcript?

The mechanisms are the binding of repressor proteins to specific mRNA sequences (specific to one transcript), the binding of proteins to specific sequences in the 3' untranslated regions of some mRNAs (specific to one transcript), modulation of the activity of initiation factors (global).

How many codons are there? Start codons? Stop codons?

There are 64 different codons; 1 start codon; 3 stop sodons

Is there translational regulation in bacteria? Why?

There is translational regulation in bacteria because they contain the subunits need to have translation.

Why are TFIID and TFIIB and a few other general transcription factors left in place upon release of the RNA-Pol II? What is achieved with that?

They act as a placeholder once RNA pol II is done transcribing; transcription achieved

What are the roles of the cap and the polyadenyl tail?

They stabilize the mRNA and help regulate translation

how many gene segments are used to code for the light chain? how many different types are there for each of these segments?

Three gene segment types V (variable): 250 segments J (joining): 4 segments C (constant) 1 segment =255 segments

downstream

Toward the 3' end of an RNA transcript (the 3' end of the DNA coding strand).

what defines the +1 position?

Transcription initiation site/transcription start site

Enhancers typically contain multiple sequence elements recognized by different transcriptional regulatory proteins

True

In reference to transcription in eukaryotic cells, the Roman number associated to TF indicates the type of RNA-Polymerase the Transcription Factor is associated to. So TFI would indicate a transcription factor associated to RNA-Pol I"

True

Splicing is also an RNA-mediated reaction (driven by a Ribozyme).

True

What do Nucleosome Remodeling Factors do? What do they need to be active?

Use energy derived from the hydrolysis of ATP to alter the contacts between DNA and histones; may act by inducing changes in the conformation of nucleosomes, affecting the ability of specific DNA sequences to interact with transcriptional regulatory proteins; can eject histones from the DNA, leaving a nucleosome-free region; can be recruited to DNA in association with either transcriptional activators or repressors, and can alter the arrangement of nucleosomes to either stimulate or inhibit transcription; Make sequences visible "push" nucleosomes to the side; Energy required-ATP consuming machine that pushes nucleosomes

What's the signal that triggers disassembly of the 48 S Pre-Initiation Complex?

When eIF2 hydrolyzes GTP to GDP, triggering a conformational change in eIF2 that results in the disruption of the 48S pre-initiation complex

What triggers GTP hydrolysis by IF2 in prokaryotes?

When the 50S ribosomal subunit associates with the complex containing the 30S ribosomal subunit, IF1, IF3, IF2 (bound to GTP), mRNA, and N-formylmethionyl tRNA, with IF2 specifically interacting with the initiator tRNA

which genes are mutated in Xeroderma Pigmentosum (XP)

XPA thru XPG (seven repair genes)

Is a ribosome a ribozyme?

Yes because the catalytic peptidyl transferase activity that links amino acids together is performed by the ribosomal RNA

A wobble base pair could be defined as

a base pair that does not follow Watson & Crick base pairing rules

Sp1 is an example of

a transcriptional activator

How about the other one?

about 30

How many genomic copies are there of rRNA genes for the ones encoded in the same transcriptional unit?

about 45-50

Poly-A tails in eukaryotic mRNAs

add stability to the mRNA, increase overall translation efficiency, and help regulate translation"

during nucleotide-excision repair, what is the role of DNA polymerase?

adds nucleotides to empty template

The process of translation of an mRNA template

always occurs in the 5' to 3' direction and results in the synthesis of a protein from the amino to the carboxy terminus

ribozyme

an enzyme in which RNA rather than protein is responsible for catalyzing the activity (Ex. RNaseP)

RNase P is _________________ and therefore _______________________

an enzyme made of protein and RNA in which the enzymatic activity resides in the RNA component; it is considered to be a ribozyme

During protein synthesis, the ________ located on the _________ interacts with the ___________ located on the __________________

anticodon, tRNA, codon, mRNA

how do DNA mutations occur

any random change in the nucleotide sequence of genomic DNA -incorporation of incorrect bases during replication -chemical changes (spontaneous, chemical exposure, radiation exposure)

in mammalian cells, transcription: a) involves making DNA from a DNA template b) involves making RNA from a DNA template c) involves making protein from an RNA template d) none of the above

b) involves making RNA from a DNA template

specific transcription factors

bind to DNA sequences that control expression of individual genes (regulation of transcription)

initiation of transcription

binding of RNA polymerase to double-stranded DNA; this step involves a transition to single-strandedness in the region of binding; RNA polymerase binds at a sequence of DNA called the promoter. Initiation is the most important step in gene expression!!! (incorporation of first 2 nucleotides in nascent RNA chain)

stages of transcription

binding, initiation, elongation, termination

eIF2-GTP - role in translational initiation?

binds to the initiator methionyl tRNA, bringing it to the ribosome.

TFIIB

bridges interaction with RNA Pol II (after TFIID)

What regulates the "recruitment" of that factor onto RNA-Pol II?

c-Myc (transcription factor)

what is silent mutation

change occurred in nucleotide sequence of genome that has no effect on type of genetic info encoded by sequence doesnt affect the functional product thats encoded

during nucleotide-excision repair, what is the role of ligase?

closes the nick that was formed

during nucleotide-excision repair, what is the role of TFIIH?

contains XPB and XPD which are helicases unwind the DNA

The TATA box and the Inr sequence are examples of

core promoters

what is a Holliday junction

cross-shaped structure that forms during genetic recombination when two double stranded DNA separate into four strands in order to exchange segments of genetic info

whats the most common type of mutation

cyclobutane rings formation between adjacent thymidines due to UV exposure

Genomic imprinting - What is it? How is it achieved?

defined as when one copy of a gene is silenced due to its parental origin. One way to silence a gene is through DNA methylation, where methyl groups are added to cytosine nucleotides in CG dinucleotides to reduce gene expression in that region. imprinted genes are dependable on the expression of whether they are inherited from the mother or from the father

what is the most efficient mechanism to fix cyclobutane rings in nature

direct reversal/photoreactivation removal of damaged bases followed by resynthesis of removed regions dna photolyase uses UV to carry out photoreactivation (not in humans or placental mammals)

what two enzymes are always involved in excision repair

dna polymerase & ligase

what is the first event during general homologous recombination? what type of enzyme would be responsible for that first event

double stranded DNA break an ENDOnuclease then digests the broken segment

what is the role of AID (activation induced [cytosine] deaminase?

drives heavy chain switch (class switch) which produces immunoglobulin genes triggers changes in sequence of variable region, in somatic hypermutation process

when does homologous recombination take place? why is it important in biological terms

during meiosis (generally) maturation of B & T cells (site specific) reverts DNA damage after replication, generates vast amounts of immunoglobulins and t-cell receptors Biological important -used to generate diversity among same species

The first GTP hydrolysis event that occurs during translational initiation in eukaryotes, which is triggered by the recognition of the start codon in the mRNA by the scanning small ribosomal subunit in association to multiple eukaryotic initiation factors, is performed by

eIF2

-What are the factors that keep the small ribosomal subunit from binding the large ribosomal subunit?

eIF3

What's the factor that activates the GTPase activity of eIF2?

eIF5

-What's the role of eEF1alpha?

elongation factor- escorts aminoacyl tRNA to the ribosome (begins elongation)

what is the role of terminal deoxynucleotide transferase

encoded by DNTT gene expressed in pre-B and pre-T lymphoid cells adds N-nucleotides to the V, D, and J exons of TCR and BCR genes during antibody gene recombination enables junctional diversity

during nucleotide-excision repair, what is the role of XPG and XPF?

endonucleases cut at both ends of bubble to remove damaged sequence

what proteins do transcription-coupled nucleotide excision repair and general genomic nucleotide excision repair share

everything after recognition RPA, TFIIH, XPD, XPB, XPF, DNA pol e/d, PCNA, and RFC

is there any other repair mechanism required after translesion DNA synthesis? why?

excision repair the error is still present after translesion polymerase has attached nucleotides

Transferrin Receptor mRNA

exemplifies mRNA degradation regulated by presence of a factor that protects the mRNA from the endonucleolytic pathway.

All eukaryotic mRNAs are polyadenylated

false

Once a protein is synthesized, there is no practical manner to control its activity; therefore, translation is considered the last layer of control of gene regulation"

false

Translation initiation requires the hydrolysis of 2 ATPs

false

what are the two main types of homologous recombination?

general homologous site-specific

what is the most important difference between homologous and site-specific recombination

general homologous- requires long regions of sequence identity site specific- requires only short stretches of sequence identity fully depends on proteins that identify regions of sequence identity and drive the recombination event also fixes double stranded breaks caused by things like ionizing radiation

what is excision repair

general means of repairing wide variety of chemical DNA alterations: Base-excision Nucleotide-excision mismatch

In eukaryotic cells, the factors that are involved in the recruitment of RNA-Pol during the transcription of most mRNAs are known as

general transcription factors

operon

genetic regulatory system found in bacteria and their viruses in which genes coding for functionally related proteins are clustered along the DNA

What is the signal used for polyadenylation?

hexanucleotide (AAUAAA in mammals) and addition to upstream and downstream (G-U rich) elements

what is the first thing that must be done before fixing any given mutation? why is this so important

identify strand that contains the mutation repairing the wrong strand will result in carrying the damaging mutation onto subsequent replication cycles

name the most important proteins involved in antigen recognition in the adaptive immune system

immunoglobulins (b-cell antibodies) and T-cells

Histone acetyl transferases could be argued to typically ___________ gene expression

increase

general transcription factors

involved in transcription from all promoters (basic transcription machinery)

what is the role of RecA

key protein involved in central steps of homologous recombination (in e. coli)

miRNAs

leads to translational inhibition followed by degradation of the mRNA targeted by the miRNA - mediated by RISC.

what are the enzymes involved in the process of site-specific recombination in lymphocytes

lymphocyte specific recombination proteins

DNA methylation occurs in ___________ located _____________________

lysine: arginine residues

rRNA processing

main event: cleavage

What's the role of eEF2?

mediates translocation

what human disease is associated w/ mutations affecting the mismatch repair mechanism?

mutations of MSH and MLH hereditary nonpolyposis colorectal cancer (HNPCC)

what are the two possible outcomes of resolving a holiday junction?

nonrecombinant heteroduplexes and recombinant heteroduplexes

whats the mechanism involved in translesion DNA synthesis

not really a mechanism to repair DNA damage its a way to bypass the damage, allowing replication to continue switches out polymerase temporarily to overcome the pyrimidine dimer block

nucleotide excision repair

oligonucleotides w/ damaged bases are removed from a molecule and replaced w/ correct bases

"In E.coli, RNA-Polymerase binds to the DNA at sequences known as"

promoters

siRNAs

provides a mechanism to target specific mRNAs to be degraded by the endonucleolytic pathway - mediated by RISC.

how is the incredible diversity of antibodies generated?

rearrangement of heavy chains, light chains, and class switching example: IgA --> IgB

what is the role of the RAG genes

recombination activating gene encodes enzymes that recombine and rearrange the genes of immunoglobulins and t-cell receptor molecules cellular expression is restricted to lymphocytes

cis-acting elements

regulatory DNA sequences that serves as a protein binding site and controls the transcription of adjacent genes (linked genes on same DNA molecule)

The enzyme responsible for transcription in eukaryotic cells

requires other factors to find the sequences that indicate where transcription should start

during nucleotide-excision repair, what is the role of XPC?

seeks the damage main protein involved in damage identification facilitates TFIIH recruitment

operator

short DNA region, adjacent to the promoter of a prokaryotic operon, that binds repressor proteins responsible for controlling the rate of transcription of the operon

In E.coli, the core RNA-Polymerase differs from the whole enzyme (the one containing all subunits) in that the core polymerase lacks the _________subunit

sigma

base-excision repair

single damaged bases are recognized and removed from a DNA molecule and replaced with correct bases

snRNPs - What are they? What do they do? Which ones are the ones that exhibit the ability to catalyze the first stage of splicing (formation of the lariat-like intermediate)?

small nuclear ribonucleoprotein particles (snRNPs), which play central roles in the splicing process; U2 and U6 are responsible for catalyzing the first stage of splicing

during nucleotide-excision repair, what is the role of RPA?

ssDNA binding protein keeps it from forming into double stranded DNA just like in replication

miRNAs affect the abundance of specific sets of mRNA in the cell mostly by ________________________

stimulating their degradation

Prokaryotic RNA polymerase

synthesizes all prokaryotic RNAs

RNA polymerase II

synthesizes protein-coding RNAs (mRNA)

RNA polymerase I

synthesizes ribosomal RNAs (28S, 18S, and 5.8S)

RNA polymerase III

synthesizes smallest ribosomal RNA (5S), transfer RNA, and some of the small RNAs involved in splicing and protein transport

In eukaryotic cells, ___________ undergo extensive modifications of their bases, affecting up to 10% of them, yielding a variety of modified nucleotides at specific positions"

tRNAs

One striking difference at the level in which transcription is initiated between prokaryotes and eukaryotes is that

the RNA-Pol are needed to initiate transcription"

TFIID

the first general transcription factor to bind the promoter, binds to the TATA box through the TATA binding protein (TBP), also includes TAFs which help recruit TFIIB

The catalytical activity associated to the ribosome is __________________. This activity resides in ______________________________

the formation of peptide bonds between the amino acids held in place by tRNAs located in the P and A sites of the ribosome; the rRNA components of the ribosome

Solving the three dimensional shape of the bacterial ribosome provided conclusive proof that

the location in the ribosome where peptide bonds are formed is made exclusively of rRNA molecules, therefore supporting the idea that peptide bond formation is a function of the rRNA components of ribosomes"

What's an Open Reading Frame?

the part of a reading frame that has the ability to be translated

what proteins do transcription-coupled nucleotide excision repair and general genomic nucleotide excision repair NOT share

the recognition proteins Transcription-coupled: RNA pol II, CSB, CSA, DDB1 General: XPA, XPC

what is the role of RuvA, RuvB, and RuvC

they're a complex of proteins that resolve the Holliday junction in e. coli

The independent chromosomal domains that delimit the range of action of enhancers are known as

topologically associating domains

upstream

towards the 5' end of an RNA transcript (the 5' end of the DNA coding strand)

Histone acetyl transferases are frequently found to act as ______________________

transcriptional coactivators

In addition to tRNAs, mRNA, and ribosomes, translation initiation in mammalian cells also requires

translation initiation factors and GTP

what is the outcome of translesion DNA synthesis

translesion dna polymerases are error prone but put the right nucleotides onto the strand complementary to the damaged strand any erros introduced by translesion polymerase (in addition to initial error) are later repaired by excision

In eukaryotic cells, eIF2 is the first GTPase to be activated during translational initiation

true

The processing of mRNAs occurs co-transcriptionally, that is, mRNAs are modified while they are still being transcribed"

true

The processing of some tRNAs requires splicing

true

how many different protein chains form an immunoglobulin

two: heavy chain protein and light chain protein each is present in two copies per immunoglobulin molecule (2 each heavy 2 each light)

Where are cis-acting sequences located?

usually within 100-200 bp upstream of the transcription start site

What's the main goal behind all the "complicated" steps involved in translational initiation?

• Dissociate the ribosomes and keep them from coming back together ahead of time • Prevent tRNAs from joining the A site ahead of time • Bring together the tRNA, mRNA, small ribosomal subunit, and detect correct start codon • Allow large ribosomal subunit to join the small one and allow entry of tRNAs into A site

What's the role of Aminoacyl-tRNA-synthetases? How many are there?

• Maintain the fidelity • Put the right amino acid in the right tRNA • Charging of the tRNA 20

Factors that activate the mRNA - what do all of them do?

• Poly-Adenyl Binding Protein (PABP) - associated with the poly(A) tail at the 3' end of the mRNA; stimulates translation by interacting with eIF4G • eIF4G (remember, G=Grouping) - binds to PABP; one of the initiation factors (other is EIF4E); in association with eIF4A and eIF4B along with initiation factor eIF4E, it brings the mRNA to the 40S ribosomal subunit, with eIF4G interacting with eIF3 • eIF4E - recognizes the 5' cap of the mRNA and forms a complex with eIF4A and eIF4G • eIF4A and eIF4B - in association with initiation factors eIF4E and eIF4G, they bring the mRNA to the 40S ribosomal subunit, with eIF4G interacting with eIF3

Differences between translational initiation in prokaryotes and eukaryotes

• Prokaryotes: o IF3 prevents two these two things to get back together o Factor One (A side) preventing rRNA from entering small subunit mediated by the Shine Delgarno sequence o IF2 ( bound to GTP) sensor; bringing mRNA will also bring the tRNA o Forms methionyl o ONLY acess to the P side Nformyl -methionyl tRNA the rest are placed in the A side o GTP event • Eukaryotes: o dissociate by eIF3 o prevent tRNA to the A side 1A (in prokaryotes is just 1) o bring everything together recognizes the cap cap binding protein eIF4 o Factor four G eIF4G - Group everything together o eIF4A + eIF4B need help from one another (scanning secondary structure) o tRNA directly methionyl Aminiacyl tRNA o start codon: scanning your message RNA

How is the start codon selected in prokaryotic cells? What's the Shine-Dalgarno sequence? Key to remember: "A Gorgeous Girl Always Gives Guys Ulcers"

• Prokaryotic don't have a cap and are polycistronic • Have the Shine-Dalgarno sequence: Initiation codons in bacterial mRNA; a ribosomal binding site in the mRNA, generally located 8 basepairs upstream of the start codon AUG. The six-base consensus sequence is AGGAGG. Lacated very short distances from the start codon