RNA+Transcription

=**Objectives**=


 * 1. List the steps in prokaryotic vs. eukarytoic mRNA synthesis** (p.221)

__Prokaryotic mRNA Synthesis__ Initiation Termination Regulation

__Eukaryotic mRNA Synthesis__ Initiation; chromatin effects, cis elements, trans factors RNA processing; capping, polyA, splicing Regulation


 * 2. Distinguish between 'sense' and 'antisense' strands of DNA and identify the template DNA strand on RNA synthesis** (p.222)

Sense strand corresponds with the mRNA sequence. It is not active for transcription and only serves as storage of DNA information.

Antisense is complementry to the mRNA sequence and is used as a template for transcription.


 * 3. List the requirements for RNA polymerase activity in terms of substrates utilized, template, and metal ions.** (p.222)

RNA polymerase activity requires ATP, GTP, CTP, UTP, an antisense template, and magnesium. RNA synthesis is driven by hydrolysis of PPi.


 * 4. Identify the direction in which RNA is synthesized** (p.222)

RNA is synthesized from 5' to 3'.


 * 5. Compare the error rates of DNA and RNA synthesis** (p.224)

RNA polymerases do not have exonuclease activity and are more error prone that DNA polymerases. High fidelity is not necessary because the large number of mRNA's produce ensure that the majority will be functional. Additionally, new mRNA is made as older mRNA is degraded, removing nonfunctional mRNA's over time. Lastly, because of the degenerate nature of the genetic code, some errors in transcription will still be translated into the correct protein product.


 * 6. Define the following and identify their roles in RNA synthesis: promoter, terminator, consensus sequence, TATA box, Pribow box, Hogness box, transcription factor, sigma factor, rho factor.** (p.225-227, 229)

Promoter - DNA sequences that are recognzied as initiation sites for RNA synthesis

Terminator - RNA sequences that signal RNA polymerase to stop syntehsizing RNA

Consensus sequence - A given DNA or RNA recognition sequence that is found in association with all the genes influenced by a particular protein. Consensus sequences make up promoters.

TATA box - a common consensus sequence in prokaryotes that makes up a promoter.

Pribow box - another name for TATA box

Hogness box - eukaryotic equivalent to the TATA box consensus sequence

Transcription factor - any protein that influences transription other than RNA polymerase

Sigma factor - A protein that allow specific promoters to be recognzed and lends specificity to the core enzyme. It decreases RNA pol affinity for DNA to allow it to slide along DNA until it hits a promoter. Sigma factor then lets of RNA pol when it reaches a promoter to allow it to bind tightly to DNA ina proceed wiht syntehsis.

Rho factor - Additional factor for termination of transcription. It bind to RNA as a hexamer and slides along the RNA, using ATP. It recognizes noncontigious termination sequences on mRNA and pulls off the newly made mRNA strand from the DNA template. IT also functions with hairpin structures.


 * 7. Identify the roles played by the following elements in prokaryotic gene expression: operator, promoter, inducer, repressor element, repressor proteins, polycistronic mRNA.** (p.231)

Operator - where repressor proteins bind to block RNA polymerase

Promoter - where RNA polymerase binds to initiate mRNA synthesis

Inducer - binds to repressor proteins, preventing repression and allowing RNA polymerase to make mRNA

Repressor Element - constituatively activated element that encodes repressor proteins

Repressor proteins - form a complex binding to the operator that prevents RNA polymerase from transcription

Polycistronic RNA - a mRNA that encodes more than one protein in sequence. More common in prokaryotes than eurkaryotes. Eukaryotes require greater control of gene expression.


 * 8. Identify the mechanism by which rafampicin, alpha-amanitin, and actinimycin D inhibit gene transcription** (p.232)

__Rafampicin__ Binds to the betal subunit of prokaryotic DNA polymerase; not toxic to humans because human polymerases are different than prokaryotic ones. Used to treat tuberculosis.

__Alpha-Amanitin__ Synthesized by the poisonous mushroom //Amanita phalloides//; inhibits both eukaryotic RNA polymerase II and II.

__Actinomycin D__ Inhibits prokaryotic and eukaryotic transcription by intercalating in DNA and blocking RNA polymerase progression.


 * 9. List the types of eurkaryotic RNA polymerases and the type of RNA that each synthesize.** (p. 233)

RNA polymerase I - 45S rRNA precursor; located in the nucleolus RNA polymerase II - hnRNA (heterogeneous nuclear), mRNA synthesis; located in the nucleoplasm RNA polyeramse III - tRNA, some snRNA synthesis; located in the nucleoplasm mtRNA polymerase- all types of RNA in mitochondria


 * 10. Describe the manner in which DNA is compacted in the chromatin structure and the role of histones.** (p.234)

DNA is wrapped around histones (2x of H2A, H2B, H3, and H4) which form nucleosomes that can compact DNA by up to 7x. Linker histones (H1) link nucleosomes together and scaffolding proteins bind to nucleosomes and form a twisted helical structure, further compacting DNA.


 * 11. Identify the characteristics of actively transcribed chromatin.** (p.235)

Chromatin structure surrounding actively transcribed genes allow greatere accessibility to regulatory proteins. The chromatin structure is less condensed and the H1 histone is frequently missing or depleted (it falls off when phosphorylated). Topoisomerases are found in association with active genes to releve torsional stress of transcription. Core histones are generally more extensively modified, frequently by acetylations which add a negative charge to the histone, making them repulsive to the negatively charged DNA. Actively transribed genes tend ot be hypomethylated.


 * 12. Identify the location on the base and the base that is primarily involved in DNA methylation in eukaryotes.** (p.235)

The C-5 carbon of cytosine can be methylated by specific methylases. Methylation occurs mainly in CG dinucleotides in eukaryotes and are called "CPG island" hotspots for methylation. Methylation blocks transcription factors and renders DNA transcriptionally inactive.

DNA methylation is responsible for genomic imprinting and is regulated by environmental effects such as growth factors or nutrients. It is important in development for proper regulation and sequence of gene activation.


 * 13. Identify the roles of the TATA box, promoter cis elements, trans-acting factors, coregulators, enhancers, and silencers in the control of eukaryotic gene expression.** (p.236, 240)

TATA box - consensus sequence on DNA that is part of the promoter where transcription factors bind and either increase or decrease affinity of RNA polymerase to the start site.

Promoter cis elements - DNA sequences where transcription factors may bind.

Trans-acting factors - factor that binds to the DNA.

Coregulators - adaptor molecules that mediate between other factors and RNA polymerase. They do not directly bind to DNA but may co-activate or co-repress a DNA binding factor.


 * 14. Elucidate the mechanism of action of tamoxifen. (p.239)**

Tamoxifen functions as an estrogen agonist and binds to an intracellular receptor which changes its conformation and translocates into the nucleus. There, the tamoxifen/receptor complex binds to the enhancer but, unlike estrogen, does not interact with the promoter and does not activate transcription of a gene.


 * 15. Identify the role of eukaryotic transcription factors in the control of gene expression and development. (p.238)**

Transcription factors can differentially control gene expression and development in different tissues by recruitment of a variety of different transcription factors. The variation of transcription factors, adaptor molecules, and promoter archetecture provide the foundation for tightly regulated gene expression necessary for proper development and function.


 * 16. Identify the main features of eukaryotic mRNA. (p.245, 246)**

Eukaryotic mRNA has a GTP that caps the 5' end and a polyadenylate tail at the 3' end; both serve to stabilize the DNA and prevent it from degradation. The 5' cap may be involved in subsequent mRNA splicing. Not all mRNA has a polyA tail - for example, mRNA for histones don't have a polyA tail, probably because histone proteins are relatively stable and do not require continuous synthesis from mRNA. hnRNA also contains introns which are non-protein coding and must be spliced out from the protein-coding exon regions to form mature mRNA for translation. Not all genes have introns though many eukaryotic genes do.


 * 17. List the steps involved in the maturation of an initial hnRNA into an mRNA. (p.248)**

(1) The 5' end is "capped," enhancing binding of the mRNA to ribosomes (2) The 3' end is usually modified by the addition of A-residues to form a "poly-A" tail (3) Portions of the hnRNA are removed in a complex process called splicing (4) The intervening sequences that are removed are called introns; the portions that are retained in the mature mRNA are the exons.


 * 18. Describe how defective mRNA splicing results in thalassemia. (p.250)**

Normal mRNA splicing of the beta-globin gene has positons on the mRNA that signal normal splice sites to remove introns from exon components. However, a mutation at one of these splice sites would inactivate splicing at the normal site but active splicing at other cryptic sites that are normally not utilized for splicing. As a result, the final spliced mRNA will differ from the normal mRNA by having either a part of an intron left in, or part of an exon left out, or both. The defect in the mRNA will result in the production of defective protein products, in this case, beta-globin chains.


 * 19. Describe how differential splicing can result in different mRNAs from the same original transcript. (p.249)**

Some genes may overlap on the mRNA transcript; alternate splicing can result in different mature mRNAs that can code for different protein products. Thus, a single mRNA transcript can produce several different splice variant protein products.


 * 1. Idenitify the reaction catalyzed by amino-acyl-tRNA synthetase** (p.258)

Amino-acyl-tRNA synthetase catalyzes the activation of amino acids for protein synthesis. There is at least one aminoacyl tRNA synthetase for each amino acid; one synthetase per tRNA. Each synthetase recognizes its own amino acid and appropriate tRNA.

Aminoacyl-tRNA synthetase catalizes two reactions:

Amino Acid + ATP --> Aminoacyl adenylate + PPi

Aminoacyl adenylate + tRNA --> Amino Acid-tRNA + AMP


 * 2. Identify the roles played by the following factors in protein synthesis: initiation factors, the Cap binding protein complex, elongation factors, and release factors.** (p.260-262)

__Initiation factors__ recruit the __cap binding protein complex__ to bind to the 5' head of the mRNA. Because Additional initiation factors with helicase activity then bind and denature mRNA secondary structures to iron out mRNA for translation. After translation is initiated, __elongation factors__ recruit specific amino acid-tRNA to bind in the A cylte of the ribosomes. Elongation factors then help mediate the translocation of the amino acid on the P-site Amino Acid-tRNA to the A-site with hydrolysis of GTP, moving forward along the mRNA. When the chain reaches a stop codon, __release factors__ come and for peptidyl transferase on the ribosome to accept H2O instead of another Amino Acid-tRNA. This results in the hydrolysis of the tRNA-peptide bond, releasing the tRNA, peptide chain, and the release factor. The ribosome then dissociates from the mRNA.


 * 3. From a diagram of tRNA, identify the positions where the amino acid is linked to it, where the anticodon is located, and what three nucleotide residues are at the 3'-end of each tRNA.** (p.257)



The amino acid is linked to the 3'-terminus at the CCA sequence. Each tRNA recognizes a spcific tRNA. The anticodon is located at the loop at bottom of the "T."


 * 4. Describe the archetecture of ribosomes and list the steps required for initiation, elongation, and termination of protein synthesis.** (p.257)

Ribosomes are composed of two subunits: a 40S and 60S in eukaryotes (and a 30S and 50S in prokaryotes). The two subunits like together such that they form interconnected tunnels for the mRNA and translated protein. The 40S subunits containsthe tRNA binding site. The 60S subunit contains the peptidyl transferase that catalizes peptide bond formation as well as a GTPase that powers the movement of mRNA during translation.

__Initiation__ (1) Aminoacyl-tRNA transferase attaches amino acids to tRNA using a two-step, ATP-dependent mechanism.

(2P) In prokaryotes, the mRNA positions itself on the 30S subunit. The sequence in the 5' leader base pairs with the 3' end of 16S rRNA on the ribosome. (3P) The sequence on the mRNA is 7-10 nucleotides upstream of the AUG or GUG start codonand is colled the Shine-Dalgarno sequence.

(2) In eukaryotes, initiation factors recruit the cap-binding protein to attach to the 5'-end of the RNA. (3) This allows binding of other initation factors with helicase-activity to denature secondary structures in mRNA (4) After this is complete, the initiation tRNA binds to the 40S subunitto form the 40S-preinitation complex. the 40S preinitiation complex slides down the RNA unstil the first initiation AUG codon is detected. (5) If the AUG codon is flanked by an optimal arrangement of nucleotides (called a Kozak consensus), translation will be initiated; otherwise, the pre-initiation complex continues to move to the next AUG. (6) If the correct Kozak sequence is found, the 60S subunit comes and binds to the small subunit and mRNA, with the initation tRNA in the P-site.

__Elongation__ (1) Elongation beings with elongation factors recruiting the next appropriate tRNA with an anticodon matching the next codon. (2) The next tRNA binds in the A-site and a new peptide bond is formed by peptidyl transferase with the ester bond of the first amino acid-tRNA breaking to attach to the amino group of the second amino acid. (3) Elongation factors mediate the translocation of the A-site tRNA to the P-site, kicking out the first tRNA and moving forward on the mRNA (4) This process continues until a stop codon is reached.

__Termination__ (1) When a stop codon is recognized in the A-site, termination factors force peptidyl transferase to accept a H2O rather than another tRNA. (2) This hydrolyzes the peptide chain from the tRNA; both of which are released. (3) The ribosomes dissociate from the mRNA.


 * 5. Match a list of inhibitors or protein biosynthesis with their mechanism of action.** (p.267)


 * Inhibitor**||**System**||**Process Affected**||
 * Streptomycin||Prokaryote||Formation of initiation complex||
 * Tretracyclin||Prokaryote||Aminoacyl tRNA binding at A-site||
 * Erythomycin||Prokaryote||Binds 50S, inihibits translocation||
 * Fusidic Acid||Prokaryote/Eukaryote||Inhibits elongation, binds eEF2/GDP||
 * Diphtheria Toxin||Eukaryote||Inactivates eEF2 by polyribosylation; not an antibiotic||
 * Puromycin||Prokaryote/Eukaryote||Aminoacyl-tRNA analog, acts as peptidyl acceptor||


 * 6. Identify what "wobble" means with respect to codon-anticodon recognition.** (p.266-267)

Wobble refers to the third codon base on the mRNA or first anticodon base on the tRNA being promiscuous about its binding, allowing different pairings to be accepted. This contributes to the degeneracy of the genetic code as a separate tRNA is not required for every codon because of wobble.

__Allowed pairings in the wobble position:__
 * First anticodon base**||**Third Codon Base**||
 * C||G||
 * A||U||
 * U||A or G||
 * G||U or C||
 * I||U, C or A||


 * 7. Identify where a Shine-Dalgarno and Kozak sequences are found and what function they perform.** (p.259-260)

Shine-Dalgarno sequences in prokaryotes and Kozak sequences in eukaryotes are sequences on mRNA that function in signal where the ribosome should bind to start translation. Prokaryotes use either AUG or GUG as start codons while eukaryotes use AUG flanked by a kozak consensus sequence.


 * 8. Identify the mechanism of action of diphtheria toxin.** (p.268)

Diphtheria toxin binds to cell membranes and is cleaved by a protease to form an active enzyme that is transported to the cytoplasm. This toxic enzyme catalyzes the ADP-ribosylation of elongation factor 2, effectivelyinactivating the elongation factor. A single molecule of diphtheria toxin is sufficient to kill a cell because it is an enzyme.


 * 9. List the general characteristics of the genetic code, include the number of nucleotides in a codon, the degeneracy of the code, and the nature of initiation and termination codons.**

The genetic code translates 3-nucleotide codons to the 20 amino acids. Because there are more 3-nucleotide codes than amino acids, some amino acids can be represented by multiple codes, leading the genetic code to be called degernate. Initation codon in prokaryotes are either AUG or GUG, coding for Met or Val. Initiation codons in eukaryotes are AUG, coding for Met. Three codons code for terminiation of translation: TAA, TAG,and TGA.


 * 10. Match the following terms with their definitions**

__Wobble__ Wobble refers to the third base in the codon and first base in the anticodon being more flexible in base pairing.

__Degeneracy__ This refers to amino acids being coded by the genetic code by multiple codons.

__Initiation codon__ AUG or GUG for Met or Val in prokaryotes. AUG for Met in eukaryotes.

__Triplet code__ All amino acids are coded as a 3 nucleotide sequence.

__Anticodon__ Three-nucleotide sequence on the tRNA that binds complementry to codons on mRNA.

__Codon__ Three-nucleotide sequence on the mRNA that binds complementry to anticodons on tRNA.

__Open reading frame__ "Reading" of every 3 non-overlapping nucleotides as coding for proteins starting from the start codon to the stop codon.

__Peptide site__ P-site of the ribosome where the initiation tRNA binds and where tRNA holding the peptide chain moves to after each peptide-bond formation step.

__Initiator tRNA__ The first tRNA holding the methionine that binds to the 40S subunit to form the 40S pre-initiation complex.

__Aminoacyl site__ A-site of the ribosome where subsequent tRNA's bind to and who which the growing peptide bond attaches to the next amino acid.

__Deletion mutation__ Mutation resulting in the loss of nucleotides.

__Insertion mutation__ Mutation resulting in the addition of nucleotides.

__Nonsense mutation__ Any change in the DNA that results in a termination codon replacing an amino acid codon.

__Null mutation__ A mutation that completely elimiates the function of a gene

__Point mutation__ Mutation involving a change in a single base pair

__Peptidyltransferase__ Enzymatic activity found on the 60S subunit of a ribosome the catalyzes the formation of peptide bonds.

__Polysomes__ mRNA + whole bunch of attached ribosomes

__Release factors__ Factor that forces peptidyltransferase to accept a H2O when it hits a stop codon, terminating translation

__Stop codon__ Sequence on the mRNA that signals the end of translation: TAA, TAG, TGA

__Shine-Dalgarno__ AUG or GUG sequence on prokaryotic mRNA that tells the ribosome where to bind.

__Frameshift mutation__ Mutation that alters the reading frame of mRNA. Also when you skip a bubble on a multiple choice exam and totally f@%k yourself.

__Cap binding protein__ Recruited by intiation factors to bind to the 5' end of the mRNA. This protein allows the subsequent binding of other initation factors that denature secondary structuers on the mRNA in preparation for translation.

__Kozak sequence__ AUG flanked by sepcific sequences that signals the ribosome where to bind.

__Aminoacyl tRNA Synthetase__ Enzyme that actives amino acids with ATP and then attaches them to the appropriate tRNA.


 * 1. Identify the covalent modifications that allow for the attachment of proteins on the inner and outer surfaces of the cell membrane.** (p.269-270)

Covalent attachment of fatty acids such as palmitoyl CoA or myristyl CoA to proteins adds a hydrophobic component that allows proteins to be associated with cell membranes.

The GPI (glycosylphosphatidyl inositol) anchors can also be attached to proteins. Unlike just hydrophobic domains on the protein, GPI anchors recognize specific targets in membrane domains (lipid rafts) that are important for cellular signaling.


 * 2. Identify the sites of linkage of N-linked and O-linked oligosaccharaides to proteins.** (p.270, 271)

__N-linked oligosaccharaides__ Single species of oligosaccharaide is transfered to an amino group of an asparagine amino acid in the growing peptide chain.

__O-linked oligosaccharaides__ Oligosaccharaide added to the -OH of serine or threonine residues


 * 3. List the potential pairs of amino acids that are the targets of cleavage by proteolytic processing enzymes .** (p.272)

Proteolytic processing enzymes typically cleave next to pairs of basic residues such as Arg-Arg or Lys-Arg.


 * 4. Describe the manner in which insulin is generated by proteolyic enzymes** (p.273)

Insulin is synthesizes as preproinsulin. Proteolytic enzymes first cleave off the NH3+ end, forming proinsulin. Next, proinsulin is cleaved at 2 sites, forming insulin, which has 2 protein components linked by two disulfide bridges.


 * 5. Identify the effect of HIV protease inhibitors.** (p.273)

HIV synthesizes a polycistronic mRNA that is coded into one big fusion protein that normally must be cleaved to the separate functional proteins by proteases. HIV protease inhibitors would prevent the cleavage of the separate proteins from the big fusion protein.


 * 6. Describe the role of ubiquitin in protein degradation.** (p.274)

Protein degradation by ubiquitin is an ATP-dependent mechanism. Basically, ubiquinating enzyme complex binds ubiquitin to the amino group on a lysine of the targeted cytosolic protein. After the first ubiquitin binds, subsequent ubiquitins bind to the ubiquitin, forming a ubiquitin-protein complex. Each ubiquitin addition uses ATP. The ubiquinated protein is then recognized by ubiquitin-dependent proteases that cuts up the protein to constituent amino acids and recycles the previously attached ubiquitin molecules.