MCB exam 3

Compare DNA pol III and RNA polymerase

-RNA polymerase does not need a primer -There are many more copies of RNA polymerase in the cell -RNA polymerase has a much slower kcat -RNA polymerase makes more errors

Prokaryotic RNA polymerase
(talk about subunits)

-the sigma subunit has a role in the regulation of transcription -the sigma subunit can be disassociated easily from the rest of the enzyme -it has 5 different subunits -the sigma subunit is responsible for accurate determination of transcription start sites

Heparin is a polyanionic polysaccharide that blocks initiation by RNA polymerase by virtue of its binding to double-stranded DNA. But heparin inhibits only when added before the onset of transcription, and not if added after transcription begins. *<b>Explain this difference</b>*

Once the transcription complex is formed, it is extremely stable and inaccessible to heparin. It forms with a Ka as high as 1012M−1.

Is RNA polymerase saturated with substrates in vivo?

It’s saturated

Describe experiments that might indicate whether RNA polymerase is operating at Vmax with respect to its nucleotide substrates.

–Add ribonucleosides to a bacterial or cell culture and determine whether the rNTP pool sizes and chain growth rates are increased. –Introduce the genes for the mutant purine and pyrimidine nucleotide synthesis enzymes into bacteria, and determine whether RNA chain growth rates are increased.

What is the order of events for RNA synthesis?

1. Polymerase finds a promoter region 2. Several DNA bases are unwound, forming a stable complex 3. A Mg+2 dependent isomerization of DNA takes place 4. Template specific rNTP’s are bound 5. Nucleophilic attack of the 3′ OH group of the first nucleotide at the a-phosphate group of the second nucleotide 5′ triphosphate occurs

Alignment of conserved sequences in promoters recognized by E. coli RNA polymerase revealed two ________ sequences upstream of the transcribed region, which are separated by similar lengths of DNA.

consensus

(t/f) In bacteria, mRNA, tRNA and rRNA are all transcribed by the same RNA polymerase.

True

(t/f) Rho-dependent termination of transcription makes use of a hexameric protein, which has RNA-DNA helicase activity.

True

Describe how RNA polymerase backtracking could function to increase the fidelity of transcription

If forward translocation slows down, the reverse process will occur more often. Just as we have seen with the 3′ to 5′ exonuclease activity of DNA polymerases, the main fidelity gain probably comes from slow extension of a mismatch (increasedKM for extension from a mismatch). The backtracking activity allows the polymerase to make a second attempt instead of just falling off. Because the probability of repeating an error is low, the second attempt to transcribe that same stretch of DNA has a high probability of transcribing it accurately.

Promoters were originally identified as consensus sequences upstream from transcriptional start sites. What additional evidence might support the assignment of these sequences as parts of promoters?

-Methods of identification of transcription start sites that look like the consensus sequences -Mutations engineered in the site under study should alter the rate of transcription of adjacent genes -RNA polymerase binds tightly to DNA in the vicinity of these consensus sequences

Steps of transcription of DNA to synthesize mRNA:

1. DNA double helix unwinds to expose the nucleotide bases 2. RNA polymerase identifies the start sequence 3. RNA polymerase adds bases that are complementary to the DNA template 4. RNA polymerase identifies the termination sequence 5. mRNA strand and RNA polymerase are released

After promoter clearance by RNA polymerase II….

a subset of factors remain at the promoter region

What does polymerase I do?

Pol I carries out the transcription of the 45S pre-rRNA transcript.

What doese polymerase III do?

Pol III is responsible for the transcription of ribosomal 5S rRNA. Pol III is responsible for the transcription of all tRNAs.

What problems are faced by chromatin during transcription? *****

–In some cases, chromatin structure rearranges at the time of replication –The action of chromatin remodeling factors is involved, which require ATP hydrolysis to allow the promotor regions to accept the complex –In some instances, protein factors can interfere with chromatin structure at specific locations, opening sensitive sites

In eukaryotes, what are all the tRNA genes transcribed by?

RNA polymerase III

Eukaryotic transcription normally takes place on a…

Chromatin template at nuclease-accessible sites

Initiation of transcription in E. Coli

–DNA melting near the transcription start site is essential for promoter recognition –The phosphodiester bonds that are formed are not stable until about 10 nucleotides have been incorporated –The sigma subunit of RNA polymerase makes strong contacts with the -10 region of promoters –The RNA polymerase contacts about 50 bp of DNA

Without adding specific regulatory proteins, how might you stimulate transcription from the gene of interest relative to the transcription of the other genes on your DNA template?

Add a dinucleotide (for example, ApC), complementary to the first two template deoxyribonucleotides, that can bypass the first nucleotide incorporation step and force transcription from that site selectively. Adding rNTPs at low concentrations will minimize initiation by rNTPs at other sites.

To make all of the complexes identical, you would like to arrest all transcriptional events at the same position on the DNA template before isolating the complex. How might you do this?

By knowing the sequence of the gene being transcribed and adding only three rNTPs, instead of all four, to the transcription mixture, with the missing nucleotide being the one that would have been inserted at the site at which you planned to arrest transcription.

The tac promoter directs transcription initiation more efficently than either the trp or lac promoters, why is this?

tac promoter more closely resembles the consensus -35 and -10 sequences than does either trp or lac. tac has 12 out of 12 identities with the consensus sequences, whereas lac has 9 and trp has 8.

RNA polymerase II

–Promoter escape occurs with the growing RNA chain exceeds seven nucleotides, triggering the release of TFIIB –The TFIIB element B-linker, in ATP-dependent process, opens up DNA before the transcription start site –TBP binds to DNA and bends it 90 degrees

In a length of DNA that contains a bacterial gene, what is the RNA-coding region

The same sequence as RNA transcript (except for having T instead of U)

What is the mechanism that recognizes the consensus sequences? (in a length of DNA containing a bacterial gene)

Recognized by the sigma subunit of RNA polymerase

What do inverted repeats produce? (in a length of DNA containing a bacterial gene)

Produces stem-loop structure in RNA transcript

What does a polyadenine sequence lead to?

Leads to an unstable RNA-DNA duplex

What are the 4 stages of bacterial transcription?

1. Promoter recognition 2. Chain initiation 3. Chain elongation 4. Chain termination

Outline the stage in bacterial transcription: Promoter recognition

First step. RNA polymerase is a holoenzyme composed of a five-subunit core enzyme and a sigma (σ) subunit. Different types of σ subunits aid in the recognition of different forms of bacterial promoters. The bacterial promoter is located immediately upstream of the starting point of transcription (identified as the +1 nucleotide of the gene). The promoter includes two short sequences, the -10 and -35 consensus sequences, which are recognized by the σ subunit.

Outline the stage in bacterial transcription: Chain initiation

second step. The RNA polymerase holoenzyme first binds loosely to the promoter sequence and then binds tightly to it to form the closed promoter complex. An open promoter complex is formed once approximately 18 bp of DNA around the -10 consensus sequence are unwound. The holoenzyme then initiates RNA synthesis at the +1 nucleotide of the template strand.

Outline the stage in bacterial transcription: Chain elongation

Third step. The RNA-coding region is the portion of the gene that is transcribed into RNA. RNA polymerase synthesizes RNA in the 5′ → 3′ direction as it moves along the template strand of DNA. The nucleotide sequence of the RNA transcript is complementary to that of the template strand and the same as that of the coding (nontemplate) strand, except that the transcript contains U instead of T.

Outline the stage in bacterial transcription: Chain termination

Fourth step. Most bacterial genes have a pair of inverted repeats and a polyadenine sequence located downstream of the RNA-coding region. Transcription of the inverted repeats produces an RNA transcript that folds into a stem-loop structure. Transcription of the polyadenine sequence produces a poly-U sequence in the RNA transcript, which facilitates release of the transcript from the DNA.

Organize the order of events as the occur during Eukaryotic transcription involving RNA polymerase II.

1. TFIID binds to the TATA box 2. TFIIB, TFIIF, and RNA polymerase II bind 3. TFIIE and TFIIH bind 4. Synthesis of the pre-mRNA begins at the +1 nucleotide 5. A 5′ cap is added to the pre-mRNA 6. Spliceosome complexes carry out intron splicing 7. A poly-tail is added to the pre-mRNA —-Transcription by RNA pol II in eukaryotes begins when TFIID recognizes and binds to the TATA box. The bound TFIID helps recruit TFIIB, TFIIF, and RNA pol II. Once those subunits of the minimal initiation complex are bound, TFIIE and TFIIH bind to form the complete initiation complex. Assembly of the complete initiation complex releases RNA pol II, which begins synthesizing the RNA transcript in the 5′ → 3′ direction. After the first 20-30 nucleotides have been synthesized, a cap consisting of a methylated guanine is added to the 5′ end of the pre-mRNA. Intron removal occurs as RNA pol II continues to elongate the pre-mRNA. When the polyadenylation signal has been transcribed, a poly-A tail is added to the 3′ end of the pre-mRNA. Polyadenylation is usually coupled with the termination of transcription.

Which of the following initially determines which DNA strand is the template strand, and therefore in which direction RNA polymerase II moves along the DNA?

the specific sequence of bases along the DNA strands

What processing takes place before a mature mRNA exits the nucleus?

—A cap consisting of a modified guanine nucleotide is added to the 5′ end of the pre-mRNA. —A poly-A tail (50-250 adenine nucleotides) is added to the 3′ end of the pre-mRNA. —Noncoding sequences called introns are spliced out by molecular complexes called spliceosomes.

Alternative splicing can….

—Produce mRNAs that are nonfunctional. —Be tissue-specific. —Contribute to temporal control of gene expression. —Produce proteins with different functions from the same gene.

What types of post-transcriptional processing occurs for bacterial tRNAs?

—Endonuclease cleavage —Exonuclease cleavage —Non-template directed addition of bases —Methylation of bases

In bacterial post-transcriptional processing of pre-rRNA what is yielded?

Mature tRNAs

Outline the sequence of events in order during the termination of transcription in Eukaryotes

1.Pol II transcribes well past the end of the gene, passing through several TTATTT regions. 2. The pre-mRNA has its signal cleaved 11-30 residues downstream of it by an endonuclease. 3. A long poly(A) tail is added by poly(A) polymerase.

During RNA processing, what is added to the 5′ end of the RNA?

Modified guanine nucleotide (5′ cap)

During RNA processing, what is added to the 3′ end of the RNA?

A long string of adenine nucleotides (a poly-A tail)

What are spliceosomes composed of?

snRNPs and other proteins

What are the RNA segments joined to one another by spliceosomes?

Exons

Where does translation occur?

In the cytoplasm (on the ribosomes, the site of translation)

Where does the degradation of bacterial mRNAs occur?

From the 5′ end

What do you know about the processing of mRNA

—Bacterial mRNA is synthesized at the nucleoid and is immediately available for translation. —The 5′ end of pre-mRNA is capped, in which a guanine nucleotide is added in the reverse orientation. —-Snurps (small nuclear ribonucleoprotein particles) complex with capped pre-mRNA.

What are the steps of mRNA production in eukaryotes in order?

1. Transcription 2. 5′ cap addition 3. Addition of poly-A tail 4. Exon splicing 5. Passage through the nuclear membrane

What is a characteristic of transcription in eukaryotes but not in prokaryotes?

Exon splicing

What is the function of the 5’mRNA cap?

It provides a safe site for ribosome binding in the cytoplasm. It is essential for recognition of the mRNA by the ribosomes in the cytoplasm.

What are some characteristics of translation?

–The anticodon region of the tRNA binds to a complementary codon region on mRNA –The resulting polypeptide is synthesized from its N terminus

Why are the three-dimensional structures of tRNAs highly conserved?

So that each can fit equally well onto the ribosome and carry out its function

Amino acids are activated by aminoacyl-tRNA synthetases–this incorporates the amino acid to the 3′ terminal end of an invariant adenosine residue on the tRNA. What do we know about this enzyme and the reaction it catalyzes?

—The amino acids react with ATP to form a mixed anhydride known as an aminoacyl adenylate. —-While still bound to the enzyme, a reaction between this anhydride and the free 3′ end of the tRNA occurs. —-There are two types of aaRS enzymes, each of which has different active sites. —In higher eukaryotes, aaRS molecules have acquired additional functionality, such as autoimmunity, control of apoptosis, and regulation for rRNA synthesis.

How do aminoacyl-tRNA synthetases ensure the fidelity of translation?

–They can sense the improper fit of an amino acid and hydrolyze the intermediate prior to attachment to the tRNA. –There is a short window of opportunity for the enzyme to hydrolyze the amino acid-tRNA bond before it is released for translation.

What are the critical components of a bacterial 70s ribosome?

—a peptidyl transferase that synthesizes peptide bonds —an aminoacyl-tRNA —three tRNA binding sites (E, P, and A) —a 30S subunit that is responsible for mRNA binding and decoding of the sequence

Which type of RNA contains an anticodon and has amino acids covalently attached as well as interprets the information from the mRNA bringing the appropriate amino acid to the ribosome?

tRNA

Which type of RNA carries the genetic information from DNA that specifies the sequence of amino acids in the new protein, and contains exons?

mRNA

Which type of rRNA is a component of ribosomes, and also the most abundant form of RNA?

rRNA

During which step of the replication/transcription/translation process does mRNA FIRST play a role?

Transcription/ RNA processing

During which step of the replication/transcription/translation process does rRNA FIRST play a role?

Translation

During which step of the replication/transcription/translation process does tRNA FIRST play a role?

Translation

What is the order of steps for the initiation of bacterial protein biosynthesis??

1.) Binding of IF1 and IF3 to the 30S subunit that promotes the disassociation of the ribosomes into the 30S and 50S subunits 2.) Initiation factor 2 (IF2) with bound GTP delivers fMet-tRNA 3.) Simultaneous binding of mRNA 4.) Reassociation of the 50S subunits 5. Release of IF3 6. The AUG initiation codon binds in the P site. 7. GTP hydrolysis to GDT, IF1, IF2, and Pi release.

What is the order of steps for the elongation of bacterial protein biosynthesis??

1.) The A-site is aligned with the mRNA codon for the next amino acid. 2.) The charged aminoacylated tRNA and elongation factor-To are escorted into the A-site 3.) The GTP is hydrolyzed by elongation factor-Tu and the aminoacylated tRNA moves into the A-site 4.) Peptide bond formation takes place and the peptidyl chain is transferred to the peptidyl-tRNA to the amino acylated tRNA, in a move catalyzed by peptidyltransferase 5.) The peptidyl-tRNA translocates from the A site to the P site 6.) The previously deacylated tRNA is released from the E site 7.) The A site now accepts the elongated peptidyl-tRNA

Amino acids are attached to tRNA by enzymes called…

Aminoacyl-tRNA synthetase

Once elongation is underway, tRNAs involved in the process occupy a series of sites on the complexed ribosome. The occupation of sites occurs in the following order.

A site, P site, E site

When a peptide bond is formed between two amino acids, one is attached to the tRNA occupying the P site and the other _______.

is attached to the tRNA occupying the A site

What catalyzes the peptide bond formation?

peptidyl transferase

mRNA

—are unstable —does not accumulate in bacteria —small amount of it is used in one translation

rRNA and tRNA

—accumulates to high steady-state levels —several molecules are involved in simultaneously translating from a single informational molecule —are metabolically stable

Ribosomal proteins have high pI values, why is this advantageous for ribosome stability?

High pI means that ribosomal proteins are basic. Hence, they are positively-charged at physiological pH, which facilitates their binding to negatively charged rRNA.

What is the minumum number of tRNA molecules that a cell must contain in order to translate all 61 sense codons?

31

Initiation in translation in bacteria

–Pairing between mRNA and rRNA places the initiation codon next to the P site in the ribosome. –fMet-tRNAfMet is the only charged tRNA that can bind to the 30S subunit on its own. –The formyl group is transferred to Met-tRNAfMet from 10-formyl-tetrahydrofolate. –Three initiation factor proteins are required to bind the mRNA and the initiator tRNA to the 30S ribosome.

Translation elongation in bacteria

GTP hydrolysis occurs after moving the aminoacyl-tRNA into the A site and before peptide bond formation

Shine-Dalgarno sequence

The bacterial Shine-Dalgarno sequence aligns the mRNA with the 16S rRNA to start translation at the correct position.

What name is given to the process in which a strand of DNA is used as a template for the manufacture of a strand of pre-mRNA?

transcription

What name is given to the process in which the information encoded in a strand of mRNA is used to construct a protein?

translation

What name is given to the process in which pre-mRNA is edited into mRNA?

RNA processing

What are polypeptides assembled from?

Amino acids

What are the steps for chain termination of bacterial protein biosynthesis

1. The completed peptidyl-tRNA is in the P site 2. The UGA (or UAA or UAG) stop codon aligns with the A site 3. Release factor 1 binds to the mRNA stop codon 4. Release factor 1 binds to the mRNA stop codon 5. Peptidyltransferase catalyzes the hydrolysis of the peptidyl-tRNA bond to release the newly synthesized peptide 6. Release factors 1 and 2 as well as GDP dissociate from the complex, followed by the release of the tRNA. 7. The 50S subunit dissociates.

The E site may not require codon recognition. Why?

The tRNA is released at the E site, so binding with the anticodon site may interfere with smooth release.

Streptomycin

interferes with normal pairing between aminoacyl-tRNAs and mRNA codon

Erythromycin

binds to 23S RNA

Chloramphenicol

blocks elongation by acting as a competitive inhibitor of the peptidyltransferase complex

Puromycin

causes premature chain termination

Tetracycline

inhibits the binding of aminoacyl-tRNAs to the ribosome

Chaperones are generally thought to facilitate protein folding.What additional functions do mitochondrial chaperones perform?

–facilitate degradation of abnormal proteins –prevent abnormal protein aggregation –participate in protein transport to organelles

Proteins synthesized on the rough endoplasmic reticulum

-signal sequence clevage -signal recognition particle -docking protein -vesicular transport

Translation, what is required for: Binding of the charged tRNA to the A site?

This step requires correct base-pairing between the codon on the mRNA and the anticodon on the tRNA.

Translation, what happens during: Formation of the new peptide bond

In the process, the polypeptide chain is transferred from the tRNA in the P site to the amino acid on the tRNA in the A site.

Translation, what happens when: Movement of the mRNA through the ribosome.

In this step, the discharged tRNA shifts to the E site (where it is released) and the tRNA carrying the growing polypeptide shifts to the P site.

Phosphofructokinase (PFK)

is an enzyme that functions in the cytoplasm during glycolysis. The targeting pathway is cytoplasm only. (Proteins that will ultimately function in the cytoplasm (PFK, for example) are translated on free cytoplasmic ribosomes and released directly into the cytoplasm.)

Insulin

a protein that regulates blood sugar levels, is secreted from specialized pancreatic cells. The targeting pathway is the ER-> Golgi -> outside the cell (Proteins that are destined for the membranes or compartments of the endomembrane system, as well as proteins that will be secreted from the cell (insulin, for example), are translated on ribosomes that are bound to the rough ER.)

Roles of RNA in protein synthesis in eukaryotes: Transcription/RNA processing

–pre-mRNA –mRNA –snRNA

Roles of RNA in protein synthesis in eukaryotes: Translation

–tRNA –rRNA

Roles of RNA in protein synthesis in eukaryotes: not used in protein synthesis

-RNA primers

Define operon

An operon is a region of DNA that codes for a series of functionally related genes under the control of the same promoter.

What molecule binds to promoters in bacteria and transcribes the coding regions of the genes?

RNA polymerase

What is allosteric regulation?

In allosteric regulation, a small molecule binds to a large protein and causes it to change its shape and activity. Allosteric regulation is an important mechanism for changing enzyme activity, as well as for changing the function of some gene repressors and activators.

Under which conditions are the lac structural genes expressed most efficiently?

No glucose, high lactose (When glucose is absent and lactose levels are high, the lac structural genes are expressed the most efficiently. Without glucose, cAMP is produced and CAP can stimulate transcription of the structural genes. In the presence of lactose, the repressor does not bind to the operator and therefore does not block transcription.)

What happens to the expression of the lacI gene if lactose is not available in the cell?

There is no change—the lacI gene is constitutively expressed. (The lacI gene is expressed regardless of the presence of lactose. Only the structural genes of the lac operon are affected by the presence or absence of lactose.)

What is the function of the lacZ gene?

This gene encodes an enzyme, b-galactosidase, which cleaves lactose into glucose and galactose. (The lacZ gene encodes b-galactosidase, a key enzyme in lactose metabolism. When lactose is present in the cell, the cell expresses lacZ and metabolizes lactose.)

Which enzyme converts ATP to cAMP?

Adenylyl cyclase (Adenylyl cyclase converts ATP to cAMP, which helps CAP bind and facilitates binding of RNA polymerase to the lac promoter.)

The operon model of the regulation of gene expression in bacteria was proposed by _____.

Jacob and monod

What are the components of a lac operon

–Lactose-utilization genes –promotor –operator (The regulatory gene, while not a part of the operon, plays a role in regulating the expression of the genes of the operon)

Regulatory proteins bind to…

the operator (Transcription is inhibited when a regulatory protein binds to the lac operon operator)

What causes dimerization

Dimerization is largely due to hydrophobic interactions between amino acid side chains of the long, central alpha helix in the N-terminal domain of each monomer, the C helix (one blue, one green).

cAMP

cAMP is bound in a pocket of the N-terminal domain of each CAP monomer. This pocket is formed between the C helix and a beta roll motif that includes beta strands1-8.

Salt bridge

A salt bridge is a combination of a hydrogen bond and an electrostatic interaction. In proteins, they occur between side chains that are oppositely charged such as aspartate or glutamate and arginine or lysine. The centers of charge must be less than 4 Å apart. Numerous electrostatic interactions are involved in cAMP binding, including: •a salt bridge between the positively charged sidechain of Arg82 and the negatively charged phosphate oxygen of cAMP •hydrogen bonds between cAMP atoms and side chain atoms of Glu72, Ser83, and Thr127 •hydrogen bonds between main chain atoms (Ser83) and cAMP •a hydrogen bond between cAMP and a Ser128, on the C helix from the opposite monomer.

Activation of transcription requires interactions between CAP and the alpha subunit of RNA polymerase. What best describes this interaction?

The carboxyl terminal domain of CAP binds to RNA polymerase via hydrogen bonds, salt bridges, and van der Waals interactions.

The alpha subunit of RNA polymerase binds to a DNA sequence centered 19 base pairs from the center of the CAP-binding site: 5′- A A A A A G – 3′. What describes the interaction between RNA polymerase and DNA?

RNA polymerase makes contact with DNA phosphate oxygens and with bases in the minor groove.

What mutation could lead to constitutive expression of the genes of the lac operon?

A mutation in the operator sequence

What is the biological role of the lac operon?

It ensures that a cell dedicates resources to the production of enzymes involved in lactose metabolism only when lactose is available in the environment.

The placement of the operator sequence between the promotor and the structural genes is critical to the proper function of the lac operon. why is this?

When the repressor binds to the operator, RNA polymerase cannot transcribe the structural genes.

The lac operon can be regulated by two distinct methods.

— The lac operon can be activated by the binding of allolactose to the repressor protein, releasing it from DNA and thereby allowing for transcription to occur. –In response to low glucose levels, cAMP is upregulated; the binding of cAMP to the cAMP receptor protein triggers the activation of the operon.

The pattern of binding of repressor and Cro to these sites determines whether the phage grows lytically or lysogenically. Describe the regulation of lytic and lysogenic cycles by bacteriophage repressor.

In the lysogenic cycle, repressor protein is produced, preventing the expression of Cro and genes for viral replication. In the lytic cycle, Cro protein is produced, preventing the expression of repressor protein; genes for viral replication are expressed. (The repressor and Cro genes lie on opposite sides of the operator region. In the lysogenic cycle, the repressor protein binds the operator (OR2 and OR1) preventing RNA polymerase from transcribing the Cro gene, and promoting repressor transcription. During lytic infection, Cro protein occupies OR3, preventing RNA polymerase from initiating transcription from the repressor promoter. RNA polymerase transcribes the Cro gene (and other genes).)

What is the sequence of the outer base pairs of the operator that are key recognition sequences for repressor binding?

A/T-C/G-A/T-A/T (The nucleotide sequences of operator DNA to which repressor protein binds consist of 14 base pairs. The outer 4 base pairs (half-site) at the ends of the operator (A/T-C/G-A/T-A/T) serve as key recognition sequences for repressor binding.)

Describe the bacteriophage repressor protein

The repressor is a dimer that binds in the major groove of DNA. (Repressor binds to operator DNA as dimers. Each monomer binds to an operator half-site (A/T-C/G-A/T-A/T). The amino-terminal domains of repressor are responsible for DNA binding and the carboxy-terminal domains (not shown) are primarily responsible for dimerization of the repressor monomers. The amino-terminal domain of each monomer is composed of five alpha-helices connected by short loops.)

What are the reasons for differential binding of Cro and cl to the same operator?

–The two α-3 helices lie closer together in Cro than in cI, which prevents as tight an interaction with DNA. –Cro makes contact with OR3 via unique Asn and Lys residues. –The cI repressor protein makes a critical specificity interaction with OR1 via a unique Ala residue.

What occurs as a result of an abundance of tryptophan in E.coli?

The 5 trp genes (TrpA – TrpE) are not transcribed. When trp is abundant, the genes involved in tryptophan synthesis are negatively regulated at the level of transcription.

What features of the trp operon are essential to the process of attenuation?

-Transcription and translation of the leader sequence occur simultaneously. -rp codons near the beginning of the leader sequence -The ability of sequences within the leader mRNA to pair with one another Because each gene encodes an enzyme involved in the trp synthetic pathway, the order in which the genes occur is likely not important in terms of the attenuation process.

Attenuator systems require….

Regulation by attenuation requires that translation of a given transcript can begin before transcription is completed. This is not possible in eukaryotes, as the two processes are spatially separated by the nuclear membrane.

E. Coli trp operon

—The small molecule ligand tryptophan binds to a repressor, activating it. —The premature termination of transcription occurs if tryptophan is abundant. —High levels of tryptophan shut down the operon.

What is an inducer?

a specific small molecule that binds to a bacterial regulatory protein and changes its shape so that it cannot bind to an operator, thus switching an operon on.

What does a repressor protein do?

A repressor is a protein that inhibits gene transcription. In prokaryotes, this protein binds to the DNA in or near the promoter.

What are regulator genes?

They code for a protein, such as a repressor, that controls the transcription of another gene or group of genes.

The lac operon is regulated through negative control, explain.

–When lactose is absent, the repressor protein is active, and transcription is turned off. —When lactose is present, the repressor protein is inactivated, and transcription is turned on

The lac operon is regulated through positive control, explain.

–When glucose is absent, another regulatory protein (CAP) binds to the promoter of the lac operon, increasing the rate of transcription if lactose is present

The genes of the ath operon are expressed only when the concentration of athelose in the bacterium is high. When glucose is absent, the bacterium needs to metabolize athelose as an energy source as much as possible. The same catabolite activator protein (CAP) involved with the lac operon interacts with the ath operon.
<b>Based on this information, how is the ath operon most likely controlled?</b>

Metabolism of the sugar athelose in this hypothetical system is controlled by an operon that exhibits both positive control and negative control. Transcription of the ath operon is turned on when athelose is present (negative control), and sped up when the bacterium runs out of glucose and must rely on athelose for energy (positive control).

Proteasomes are?

Enzyme complexes that break down protein

The nuclear membrane’s role in the regulation of gene expression involves…?

regulating the transport of mRNA to the cytoplasm

What is the function of a spliceosome?

RNA processing

What is the role of protein-phosphorylating enzymes’?

regulation of gene expression involving protein activation

What term describes the DNA-protein complexes that look like beads on a string?

Nucleosome

Which regulatory elements are composed of DNA sequences?

—promoter-proximal elements —Silencers —Enhancers

What do regulatory transcription factors bind to?

promoter-proximal elements and enhancers

What do basal transcription factors bind to?

The promoter.

What regulatory DNA sequence might be located thousands of nucleotides away from the transcription start site of a gene?

Enhancers They can function thousands of nucleotides away from the promoter and transcription start site.

What event in transcription initiation likely occurs last?

RNA polymerase binding to the promoter gene. RNA polymerase is recruited only when other transcription factors, including TBP, are assembled at the promoter.

How would one be able to disrupt the interaction between histone proteins and negatively charged DNA?

The positive charge on histone proteins allows them to interact tightly with negatively charged DNA, thus inhibiting transcription. *To disrupt this interaction, the histone proteins would have to be made more negatively charged*

Modification of the chromatin structure in Eukaryotes

–Methylation of histone tails in chromatin can promote condensation of the chromatin. —Some forms of chromatin modification can be passed on to future generations of cells. —Acetylation of histone tails is a reversible process. —Acetylation of histone tails in chromatin allows access to DNA for transcription. —DNA is not transcribed when chromatin is packaged tightly in a condensed form. ((One of the mechanisms by which eukaryotes regulate gene expression is through modifications to chromatin structure. When chromatin is condensed, DNA is not accessible for transcription. Acetylation of histone tails reduces the attraction between neighboring nucleosomes, causing chromatin to assume a looser structure and allowing access to the DNA for transcription. If the histone tails undergo deacetylation, chromatin can recondense, once again making DNA inaccessible for transcription. Changes in chromatin structure may be passed on to future generations of cells in a type of inheritance called epigenetic inheritance..))

In eukaryotic transcription what is the function of a histone chaperone?

Histone chaperones facilitate the reassociation of histones with DNA, to reform chromatin after DNA is transcribed.

In eukaryotic transcription what is the function of a chromatin remodeling complex?

Chromatin remodeling complexes clear nucleosomes away from promoter regions, so that transcription initiation complexes can form and function.

In eukaryotic transcription what is the function of mediator?

Mediator connects upstream regulatory proteins, bound at enhancer or inhibitory sites, to DNA pol II at the transcriptional start site.

Why does histone deacetylase action tend to repress transcription?

Removal of acetyl groups from lysine residues in histones yields positively charged free amino groups, which form ionic interactions with negatively charged DNA phosphates.

Why are antibiotics that inhibit translation useful?

the translational machinery of eukaryotes is sufficiently different to that of bacteria.

Termination of translation require __________ and direct interaction of a release factor with a termination codon.

GTP hydrolysis

Describe the transcriptional regulation of the lac operon

On in the presence of lactose and the absence of glucose

Describe attenuation of the trp operon

A transcription termination sequence forms in the leader mRNA because the ribosome reaches a translation stop codon if tryptophan levels are high.

Lac operon

–The operon is inducible; that is, its expression is condition dependent –The operon consists of three regulatory genes: LacZ (yielding β-galactosidase), lacA (yielding thiogalactoside transacetylase), and lacY (yielding β-galactoside permease).

lacZ

β-galactosidase

lacl

repressor

lacY

permease

CRP-binding site

cAMP binding protein

lac A

transacetylase

lactose repressor

—-The repressor is a negative regulator because when it is active, it inhibits transcription of the lac operon. —– The repressor can bind essentially anywhere on DNA, sliding along its length in search of its operator binding site. —- The repressor has a very low K d value.

(t/f) The λ repressor can act as a transcriptional activator as well as a repressor.

true

(t/f) Catabolite or glucose repression of the lac operon is a regulatory system, which depends on the levels of cAMP in the cell.

true

(t/f) The helix-turn-helix is a common protein motif that is able to interact with DNA in the major grooves by both electrostatic and specific interactions between amino acids of the helix and the bases of the DNA.

true

Is attenuation likely to be involved in eukaryotic gene regulation?

No, because transcription and translation in eukaryotes is separated.

What do studies on attenuation tell us about mechanisms of transcription termination in bacteria?

The formation of a stem-loop structure followed by an uracil-rich sequence is an essential structural feature of a transcriptional termination site.

Replication: goes from _______ to _______ and happens in the ___________.

DNA –>DNA in the nucleus

Transcription: goes from _______ to _______ and happens in the ___________.

DNA–>RNA in the nucleus

Splicing: goes from _______ to _______ and happens in the ___________.

Pre-mRNA –>mRNA in the nucleus

Translation: goes from _______ to _______ and happens in the ___________.

mRNA –> protein in the cytosol

Topoisomerase

process: both replication and transcription (not translation)

Helicase

process: both replication and transcription (not translation)

Primase

process: only replication

DNA polymerase

process: only replication

Okazaki fragment

process: only replication

RNA polyerase

process: only transcription

Promoter

process: only transcription

Ribosome

process: only translation

Amino acids are added to the growing polypeptide chain via _________ that have been "charged" (or prepared) with an amino acid.

tRNA

The ribosome uses a/an ______ template to direct protein synthesis.

mRNA

Translation proceeds from the ____-terminus to the ___-terminus of a protein

N-terminus, C-terminus

How many subunits is the ribosome comprised of?

2

In gel electrophoresis DNA molecules migrate from _____ to _____ ends of the gel.

negative…..positive An electrical current is generated across the gel, and DNA molecules migrate from the negative end toward the positive end.

Based on size, how far do DNA molecules move in gel electrophoresis?

The shorter the DNA molecule, the farther it moves

GFP fusion technology would be best used for what?

localizing the compartment where Golgi proteins are found

GFP fusion technology is a good technique to use for….

–for soluble proteins found in the cytosol –for transmembrane proteins with cytoplasmic domains –for proteins restricted to the nucleus

What is important in cloning the GFP cDNA into the vector?

The GFP cDNA is in the same reading frame as the target protein.

Cells expressing the GFP fusion protein will show florescence __________.

where the GFP fusion protein is localized in the cell

What is a potential limitation of GFP?

Short wavelengths of excitation light can damage the cell’s DNA.

X-Gal is included in the growth medium on which cells transformed with bacterial plasmids are grown. The reason X-Gal is included is to _______.

identify bacteria that contain a recombinant plasmid Colonies produced from cells containing a recombinant plasmid are white, whereas colonies from cells containing a nonrecombinant plasmid are blue.

Which part of a DNA nucleotide contains a negative charge?

The phosphate group of a nucleotide, contains a negative charge.

What is the method called where a large sample of DNA is made from a small sample of DNA.

PCR method. PCR is a method for amplifying DNA.

If you hook up the wires to the gel electrophoresis chamber backwards, with the positive pole at the top, and the negative pole at the bottom. If i turn on the power, what will happen to the DNA?

The DNA will move up the gel. DNA normally moves down through a gel due to the attraction of opposite charges. In this case, the DNA would move up the gel, toward the positive pole at the top.

Initiation of transcription in bacteria

–Promoters are sites on DNA that signal the positions where RNA polymerase should bind. –There are consensus sequences within the promoter sites that are conserved. –A biological technique known as footprinting can be used to identify the upstream DNA binding sites for RNA polymerase.

In a microarray analysis used to compare sporulating and nonsporulating yeast, a spot that appears yellow at a given time point is associated with a gene that is _______.

–is expressed roughly equally in both sporulating and nonsporulating yeast Yellow signal is the result of roughly equal parts red and green signals combined. This indicates roughly equal expression levels of the corresponding gene in both yeast samples.

DNA microarrays address questions related to….

…

What is the first step in chromatin immunoprecipitation?

Cross-linking with formaldehyde

Not long ago investigators were surprised to learn that more than 95% of a mammalian genome is transcribed even though less than 2% encodes proteins.
<b>What kind of evidence could be used to determine the percentage of the genome that is transcribed?</b>

–microarray data — reverse transcription + deep sequencing

Briefly describe the operation of chromatin immunoprecipitation

Chromatin was treated with formaldehyde to covalently crosslink proteins to DNA. The crosslinked chromatin was disrupted by sonic oscillation. The crosslinked fragments were immunoprecipitated with antibody to histone H2A.Z, and precipitated complexes were subjected to DNA sequence determination.

Identify the domains of Argonaute

Middle domain, Piwi domain, N-terminal domain, PAZ domain The N-terminal domain includes a "stalk" that helps support the Paz domain. The Middle, Piwi, and N-terminal domains together form the "crescent" of the Argonaute protein. The yellow structure is an inter-domain connector that clasps all four domains.

Which Argonaute domain contains the active site?

The piwi domain

Why is the genetic code said to be "degenerate?"

Each amino acid can be specified by more than one codon.

Types if mutations: transitions

pyrimidine to pyrimidine (t—c) purine to purine (a—g)

Start codon

AUG school starts in august

Stop codons

UGA, UAA, UAG

Types of mutations: transversions

pyrimidine to purine (t — a OR g) purine to pyrimidine (a—g OR c)

Having a mutation in which position is most impactful?

1st or second

What contains start and stop signals for protein synthesis?

mRNA

in bacteria, what binds to the initiator codon?

formyl-met-tRNA (both at AUG but with prokaryotes, the fMet is needed to bind to AUG)

In eukaryotes, the AUG nearest the ______ is the initiator codon.

5′ cap

In prokaryotes, the AUG nearest the _____ is the initiator codon

Purine-rich, shine dalgarno sequence.