MCB Unit 3: Gene Expression
(-)ssRNA
"negative-strand sense RNA;" also known as anti-sense; RNA that is not ready to be translated
(+)ssRNA
"positive-strand sense RNA;" RNA that is ready to be translated
CAAT box
-(GGCCAATCT) essential eukaryotic promoter sequence involved in binding transcription factors -Located between -40 and -150 base pairs upstream from the starter site. Can be located on either strand of DNA.
TATA box
-A DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex. -Typically found between positions -30 and -100. (Note that it is similar to the structure resembled in prokaryotes at the -10 sequence, but is farther from the start site in eukaryotes) -A mutation of a single base in this sequence markedly impairs promoter activity, which shows that the precise sequence AND a high concentration of AT pairs is essential to this structure. -Often paired with an initiator element
downstream core promoter element (DPE)
-A common promoter element in eukaryotes that is found in conjunction with initiator element in transcripts that lack the TATA box. -Located downstream of the starter site, between positions +28 and +32.
initiator element (Inr)
-A pyrimidine-rich sequence that spans the transcription start site -Located between positions -3 and +5. -This sequence defines the start site because the other promoter elements are at variable distances for the start site. Its presence at the start site increases transcriptional activity. -Often paired with a TATA box -In conjunction with DPE in sequences that lack TATA box
RNase P
-A ribonuclease molecule involved in generating tRNA molecules from larger precursor RNA molecules -Removes nucleotides from the 5' end of the precursor of pre-tRNA
ribosome
-A ribonucleoprotein particle made of two subunits (60S and 40S). It is the site of protein synthesis. It contains 3 tRNA-binding sites that bridge the 30S and 50S subunits (The A site binds the incoming tRNA, the P site binds the tRNA with the growing peptide chain, and the E site binds the uncharged tRNA before it leaves the structure) -2/3 of the mass is made up of RNA -In E.Coli - sediments at 70S and composed of 2 subunits: a large 50S subunit (23S rRNA, 5S rRNA, 28S rRNA, 5S rRNA) a smaller 30S subunit (16S rRNA and 18S rRNA)
Class II aminoacyl tRNA synthetases
-Acylate the 3'-hydroxyl group. -Are diemeric. -Includes enzymes for the following 10 amino acids Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, and Thr
Class I aminoacyl-tRNA synthetases
-Bind a tRNA at the acceptor stem and the anticodon loop -Acylate the 2'-hydroxyl group of the terminal adenosine -Are monomeric -Includes enzymes for the following 10 amino acids Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, and Val
transport vesicles
-Carry cargo proteins to their final destination. 1) Cargo receptor bind to the newly synthesized protein in the ER, which assists the budding of the membrane and the formation of transport vesicles. 2) Coat proteins bind to the cytoplasmic side of the vesicle to complete budding. 3) Transport vesicles bud off the ER and carry newly synthesized proteins to the golgi complex. 4) Fusion of the transport vesicles with the golgi membranes allows O-glycosylation in the golgi complex 5) From the golgi, transport vesicles deliver proteins to their designated destination 6) Integral membrane proteins of the vesicle, called v-SNAREs, bind to specific proteins in the target membrane called t-SNAREs 7) Membrane fusion occurs, delivering the protein to its destination.
enhancer sequences
-Cis-acting elements that have no promoter activity, but can stimulate the effectiveness of promoters, even when located thousands of nucleotides away from the start site of transcription. -They can be located upstream, downstream, or even in the middle of a transcribed gene. -They operate in conjunction with specific enhancer-binding proteins (activator proteins)
GC box
-In eukaryotes the region in the promoter containing 5' GGGCGG 3' sequence which is a binding site for transcriptional regulatory proteins -Located between -40 and -150 base pairs upstream from the starter site. Can be located on either strand of DNA. -Common in genes that are continuously expressed
pre-tRNA
-The primary transcript, which is cleaved to form transfer RNAs. -Contains a leader sequence (nucleotides from the 5' end of the precursor that gets removed by RNase P) -Contains an intron that is removed by endonuclease and the resulting two products are joined together by a ligase, which exposes the anticodon. The CCA-adding enzyme adds nucleotides to the 3' end, creating an amino acid attachment site.
RNA polymerase II
-a type of RNA polymerase located in the nucleoplasm, where it synthesizes the precursors of mRNA and several small RNA molecules (such as those of the splicing apparatus and many of the precursors to the small regulatory RNAs). -This RNA polymerase is very sensitive to the toxin alpha-Amanitin, as they bind very tightly (due to a Kd of 10 nM) and blocks elongation phase of RNA synthesis (alpha-Amanitin inhibits this polymerase). -In comparison to other eukaryotic polymerases, it has a unique domain called the carboxyl-terminal domain (CTD) that plays an important regulatory role.
spliceosome
A large complex made up of small nuclear ribonucleoprotein particles (snRNPs), small nuclear RNAs (snRNAs) and some associated proteins that splices RNA by interacting with the ends of an RNA intron, releasing the intron and joining the two adjacent exons. It begins with the cleavage of the phosphodiester linkage between the upstream exon (exon 1) and the 5' end of the intron. The attacking group (2'-OH of an adenlyate residue at branch site) attacks the carbonyl group of the ester linkage of the peptidyl-RNA, forming an 8-membered transition state. The transition state collapses to form the peptide bond and a deacylated tRNA.
Group II intron
A large, self-splicing ribozyme that catalyzes its own excision from an mRNA transcript as a lariat structure. It utilizes a adenosine nucleotide or the attacking 2'-OH to initiate the reaction.
Group I intron
A large, self-splicing ribozyme that catalyzes its own excision from an mRNA, tRNA, or rRNA transcript in a reaction that requires a guanosine nucleotide or nucleoside to initiate the reaction.
sedimentation coefficient
A measure of the rate of movement due to centrifugal force. -Usually expressed in Svedberg units (S), which is equal to 10^-13s. The smaller the S value, the more slowly a molecule movies in a centrifugal field. -The velocity of a particle depends in part of its mass. A more massive particle sediments more rapidly than does a less massive particle of the same shape and density.
Hammerhead ribozyme
A ribozyme that utilizes guide RNA to cleave substrate RNA at NUH (where N=any nucleotide, U=uracil, and H= any nucleotide but guanine) triplets 3' to the H Consists of a conserved catalytic core.
stem-loop structure
A secondary structure of RNA containing internal GC base pairing such that a hairpin loop forms during its transcription, which serves as a mechanism to terminate continued transcription. It is formed from single-stranded DNA and RNA molecules. Elaborate structures may form as a result of this and can be stabilized by Mg2+ ions.
consensus sequence
A sequence of DNA, RNA, or protein that represents aligned, related sequences. It consists of the most common nucleotides or amino acid residues at each position.
transcription activator-like effector nucleases (TALENs)
A sequence-specific nuclease and domains consisting of 34 amino acids per repeat, two of which specify its nucleotide binding partner. They can be designed to uniquely recognize extended oligonucleotide sequences. The mutation of these residues within an array of repeats enables the recognition of a vast number of possible DNA target sequences with a high degree of specificity.
codon
A specific sequence of three adjacent bases on a strand of mRNA that provides genetic code information for a particular amino acid and can be recognized by a 3-base complementary sequence in tRNA
TFIIF
A transcription factor for RNA polymerase II in eukaryotes that assists the RNA polymerase II to bind to the promoter and TFIIB, and helps attract TFIIE and TFIIH
TFIIH
A transcription factor for RNA polymerase II in eukaryotes that contains a DNA helicase to unwind DNA and activates RNA polymerase by phosphorylation of the CTD.
TFIIB
A transcription factor for RNA polymerase II in eukaryotes that recognizes BRE element in promoters and recruits RNA polymerase II and TFIIF to the starter site for selection.
TFIIE
A transcription factor for RNA polymerase II in eukaryotes that recruits TFIIH, modulates TFIIH helicases, ATPase, and kinase activities
TFIID-TAFs (TBP-associated factors)
A transcription factor for RNA polymerase II in eukaryotes. It is a core promoter that recognizes non-TATA sequences such as initiator element and DPE, has positive and negative regulatory functions, and contains HAT activity.
TFIID-TBP (TATA-box-binding protein)
A transcription factor for RNA polymerase II in eukaryotes. It is the TATA binding protein that recognizes core promoter and recruits TFIIB. It is also a protein complex that initiates the assembly of the active transcription complex in genes containing the TATA box.
TFIIA
A transcription factor for RNA polymerase II in eukaryotes. Stabilizes binding of TFIID and TBP to the promoter
eukaryotic promoters
AKA cis-acting elements; they bind to proteins called trans-acting elements or transcription factors to regulate polymerase activity. Each RNA polymerase has a distinct type of this that is recognized by distinct transcription factors. -Consists of conserved sequences that attract polymerases to the start site. However, it differs from prokaryotic versions as they differ in sequence and position.
translocation
After nucleotide addition, the RNA-DNA hybrid can translocate through the RNA polymerase, bringing a new DNA base into position to base-pair with an incoming nucleoside triphosphate.
B. a subset of factors remain at the promoter region.
After promoter clearance by RNA polymerase II: A. all associated factors remain bound to the polymerase for elongation. B. a subset of factors remain at the promoter region. C. no further elongation factors are acquired by the polymerase. D. no further chromatin remodeling is required. E. transcription continues until the polymerase reaches the poly (A) tail.
Heat-shock transcription factor (HSTF)
An additional factor that binds to upstream regulatory sites in Drosophila, specifically by binding to the following sequence: 5′-CNNGAANNTCCNNG-3′. This enhances the expression of certain genes after an increase in temperature.
aminoacyl-transfer RNA synthetase
An enzyme that links amino acids and nucleic acids, which allows it to translate the genetic code. It catalyzes the activation of amino acids by forming an ester linkage between the carboxyl group of the amino acid and either the 2' or 3' hydroxyl group of the terminal adenosine of the tRNA. The overall reaction utilizes the equivalent of 2 ATP.
Gene therapy
An experimental technology that allows researchers to provide functioning copes of genes to cells with disease-causing versions of those genes. The therapeutic genes can be delivered inside cells in a variety of ways. The most common delivery method uses viral vectors, which are modified viruses in which harmful portions of the virus genome are replaced with the therapeutic gene. When a viral vector gets introduced to the body, it infects target cells and releases its genome, including the therapeutic gene. The cell's machinery then produces functional protein from the introduced gene. Ex) Severe Combined Immunodeficient (SCID), which compromises T and B cell production in newborns, has been treated by infecting cells with a function version of the mutated enzyme, adenosine deaminase. Ex) Leber congential amaurosis (LCA), in which the cells of the retina are effected and cause extreme far-sightedness or blindness at birth, treated by targeting mutations in gene RPE65
GTP
Both the SRP54 and the SRα subunits of SR must bind to _______ to facilitate the formation of the SRP-SR complex.
mRNA precursors are processed and spliced in the nucleus before being transported to the cytoplasm for translation.
Briefly describe the transcription/translation in eukaryotes
The primary transcript serves as mRNA and is used immediately as the template for protein synthesis
Briefly describe the transcription/translation in prokaryotes.
positive
COVID is a virus that has a positive- or negative- strand sense RNA genome?
exons
Coding segments of eukaryotic genes. Many of these segments encode protein domains and structural/functional units of proteins found in DNA. New proteins can evolve from shuffling or duplication of these segments.
cDNA
Complementary DNA; double-stranded DNA molecule produced synthetically by reverse trascribing mRNA (a retroviral enzyme). Because of eukaryotic mRNA splicing, cDNA contains no introns.
1) an RNA copy is made that encodes directions for protein synthesis . This mRNA can be thought of as a photocopy of the original information (can be made in multiple copies, used, then disposed of). 2) The information in mRNA is translated to synthesize functional proteins and other types of RNA can exist to facilitate this translation.
DNA is often thought of as archival information that is stored and manipulated judiciously to minimize damage and mutations. Describe the two steps of how this works.
1) U1 snRNP initiates the splicing process by binding at the 5′ splice site. U1 snRNA contains a conserved six-nucleotide sequence that base-pairs to the 5′ splice site of the pre-mRNA. 2) Then, U2 snRNP binds at the branch site. 3) Binding of U4-U5-U6 tri-snRNP completes spliceosome formation. *The U2 snRNA and the U6 snRNA, components of their respective snRNPs, are the actual catalysts of splicing. The structure of the catalytic center from the spliceosome includes two key magnesium ions bound to the U6 RNA. The catalytic snRNAs facilitate transesterification reactions that remove the introns and join the exons.
Describe the 3-step process of spliceosome assembly and behavior.
1. The 5' end is modified by the addition of the 5' cap (which is a 7-methylguanosine) in which a GTP is added to the precursor in an unusual 5'—5' linkage. After linkage, 1 or more methyl groups are added. 2. Noncoding stretches of RNA (introns) are added and the products ligated to form mature mRNA. 3. The 3' end is cleaved by a specific endonuclease (downstream of AAUAAA sequence), and a stretch of polyadenylate is added by poly(A) polymerase to form the poly(A) tail on the 3' end, about 250 nucleotides long, which terminates transcription. 4. Mature mRNA is then exported out through the nuclear pore complexes to the cytosol for translation to occur.
Describe the 4 steps in which mRNA is modified, spliced, and exported out of the nucleus.
1) RNA polymerase III is recruited to the DNA by transcription factors. 2) The transcription bubble forms. 3) The CTD is phosphorylated during initiation and the polymerase escapes the promoter. 4) Transcription elongation is aided by elongation factors after TFIIE and TFIIH dissociate. 5) Elongation factors dissociate and the CTD is dephosphorylated as transcription terminates, a process facilitated by termination factors.
Describe the 5 steps of eukaryotic transcription by RNA polymerase II.
1) Begins with the cleavage of the phosphodiester linkage between the upstream exon (exon 1) and the 5' end of the intron. The attacking group is the 2'-OH of an adenylate residue at the branch site. (transesterification) 2) This process generates a branch and a lariate intermediate. 3) The 3'-OH of exon 1 attacks the phosphodiester linkage between the intron and exon 2, joining the two exons together via another transesterification reaction.
Describe the process of splicing of introns in pre-mRNA using transesterification.
the production of multiple different proteins from a single gene
Differential mRNA processing in eukaryotes, such as in using alternative splicing sites or alternative poly A sites, may result in:
Eukaryotic genes
Discontinuous genes with exons (coding regions) with interruptions of introns (non-coding regions) Note: average human gene has 8 introns and some have more than 100.
1. Search for and bind to promoter sequences, which are initiation sites for RNA synthesis 2. Unwind a short stretch of the double helix DNA to reveal the bases to be transcribed 3. Select the ribonucleoside triphosphate that corresponds to the DNA template and catalyze the formation of a phosphodiester linkage 4. Detect termination signals that halt transcription 5. Interact with other proteins that regulate the process of transcription
During transcription, what 5 steps does RNA polymerase undergo? **Note: This process is relevant in both prokaryotes and eukaryotes
RNA polymerase
Enzyme that initiates and elongates the RNA product with the chain growing in the 5' to 3' direction. Catalyzes the synthesis of RNA from a DNA template via transcription. Has 3 requirements: 1) DNA template 2) 4 activated ribonucleoside triphosphates 3) Divalent metal ions (typically Mg2+ or Mn2+)
RNA processing
Eukaryotes very extensively process nascent RNA destined to become mRNA. This processing includes modifications to both ends and splicing out segments of the primary transcript.
hnRNA
Heterogeneous nuclear RNA; the primary transcript made in eukaryotes before splicing; acts as a precursor and intermediate of mature mRNAs and other RNAs
Each aminoacyl-tRNA synthetase takes advantage of the properties of its amino acid substrate. Threonine is especially similar to two other amino acids - valine and serine. The threonyl-tRNA synthetase avoids coupling these incorrect amino acids because the structure of the amino acid binding site of threonyl-tRNA synthetase contains a zinc ion, bound to the enzyme by two histidine residues and one cysteine residue. The remaining coordination sites are available for substrate binding. Threonine binds to the zinc ion through its amino group and its side chain hydroxyl group, which is further recognized by an aspartate residue that hydrogen bonds to it. In valine, the methyl group takes place of the hydroxyl group.
How does aminacyl-tRNA synthetase possess a high level of specificity?
with the N-terminal domains, as the polypeptide exits the ribosome.
How does the folding of a 3-D structure of a protein begin?
E.coli
In _________ cells, the same RNA polymerase synthesizes all three types of cellular RNA (mRNA, tRNA, and rRNA)
cap structure, poly A tail
In eukaryotes, after transcription, the 5' of mRNA is modified by the attachment of a ______ _________ while the 3' end acquires a _________ _ ________.
complex transcriptional regulation
Like bacteria, eukaryotes rely on conserved sequences in DNA to regulate the initiation of transcription. The difference is that eukaryotes use a variety of types of promoter elements, each identified by its own conserved sequence. Not all possible types will be present together in the same promoter. In eukaryotes, elements that regulate transcription can be found at a variety of locations in DNA, upstream or downstream of the start site and sometimes at distances much farther from the start site than in prokaryotes.
TFIID --> TFIIA ---> TFIIB --> TFIIF in complex with RNA polymerase II --> TFIIE and TFIIH bind sequentially (but TFIIE must bind first because it regulates TFIIH)--> kinase and helicase activity result in phosphorylation of CTD and unwinding of DNA, aka the pre-initiation complex (PIC)
List the order in which transcription factors bind to generate the basal transcription complex.
pre-initiation complex (PIC)
Macromolecular assembly of different transcription factors at the promoter for the start of transcription. Contains RNA polymerase II and transcription factors D, A, B, F, E, and H.
Transesterification
Moving one phosphodiester bond from one point to another. Occurs in forming pterm-117eptide bonds in RNA translation forming proteins and spliceosomes.
introns
Noncoding segments of nucleic acid that lie between coding sequences in eukaryotic genes. Sizes range from 50-100,000 nucleotides. They were initially detected by electron microscopy studies. -Almost always begin with a GU and end in an AG with a branch site in the middle
1. The final mature RNA is cleaved from a larger precursor molecule and may have other modifications. 2. Many tRNA transcripts lack the CCA sequence at the 3' end of the strand. These nucleotides are added post-transcriptionally. 3. The bases and riboses of tRNA and rRNA are modified, for instance, by the attachment of methyl groups.
Precursors of tRNA and rRNA are cleaved and chemically modified after transcription. Describe 3 ways in which they can be modified.
DNA template
RNA is synthesized using a _______ _________.
slower; more This is because the elongation of RNA occurs at a rate of ~50 nucleotides per second while DNA replication occurs at a rate of ~1000 nucleotides per second. RNA polymerase makes a mistake every ~10^4 nucleotides, while DNA polymerase with proofreading makes a mistake every ~10^9 nucleotides.
RNA synthesis is (slower/faster) and (less/more) error-prone than DNA synthesis.
regulatory RNAs (siRNA, miRNA, etc.)
RNA that contributes to regulation of transcription and translation
Rho protein
RNA-binding hexameric helicase protein that has 6 subunits and helicase activity (that occurs via ATP hydrolysis) that is responsible for terminating transcription. This occurs by the protein crawling up RNA that it is synthesizing to reach the DNA/RNA hybrid and uses its helicase activity to unwind the hybrid to disassemble the polymerase and terminate transcription.
Ribonuclease P
RNase P; a ribozyme/ribonucleoprotein endonuclease that processes the 5" ends of precursor tRNAs. It binds to the 5' end and cleaves tRNA.
The single-strand RNA genome of a retrovirus is converted into a double-helical DNA by reverse transcriptase, which is an enzyme brought into the cell by the infecting virus particle. Reverse transcriptase possesses several activities and catalyzes the synthesis of a complementary DNA strand, the digestion of RNA, and the subsequent synthesis of the DNA strand. Viral RNA --> synthesis of DNA complementary to RNA --> DNA-RNA hybrid --> digestion of RNA --> DNA transcript of viral RNA --> synthesis of second strand of DNA --> double helical viral DNA
Retroviruses, such as HIV-1, are made through actions of reverse transcriptase. Define the reverse transcriptase process.
endoplasmic reticulum
Ribosomes bound to the ________ __________ manufacture secretory and membrane proteins.
strong promoters
Sequences that bind RNA polymerase strongly. The gene produces lots of mRNA when turned "on". Can be identified by being nearly similar to the consensus sequence.
weak promoters
Sequences that bind RNA polymerase weakly. The gene produces relatively low amounts of mRNA when turned "on". Can be identified by a lot of differences between the promoter and the consensus sequence.
2
Since Fokl functions as a dimer, how many ZFN/TALEN constructs are necessary to generate a double-stranded break?
C. snRNA
Spliceosomes are primarily composed of what types of RNA? A. miRNA B. snoRNA C. snRNA D. rRNA E. tRNA
true
T or F: ALL RNA synthesized by RNA polymerase II has a 5' cap and no consensus sequence is required.
True
T or F: mRNA undergoes little to no modification after synthesis in bacteria, but the same cannot be said for tRNA and rRNA
D. RNA polymerase II
The "death cap" mushroom Amanita phalloides is poisonous because it contains alpha-amanitin, which inhibits: a. DNA polymerase I b. DNA polymerase III c. RNA polymerase I d. RNA polymerase II e. RNA polymerase III
purine (adenine or guanine)
The 5′ end of the primary transcript in pre-mRNA in eukaryotes is usually a ___________.
backtracking
The RNA-DNA hybrid can occasionally backtrack within the RNA polymerase. In the backtracked position, hydrolysis can take place, producing a configuration equivalent to that after translocation. Backtracking is more likely if a mismatched base is added, facilitating proofreading.
exon skipping
The directed removal of exons during mRNA processing to restore the reading frame of the mature mRNA to make a shortened, but functional protein. It is a technology that changes how primary RNA transcript of a gene with a disease-causing mutation is spliced by removing the mutation from the resulting mRNA. A short segment of single-stranded RNA (antisense RNA) is recognized by the spliceosome and binds to a sequence of the primary RNA transcript for a gene that is normally recognized by the cell's splicing machinery. When the antisense RNA is introduced the the cell, it binds to the target sequence in the primary RNA transcript, causing the splicing machinery to skip over a segment of the transcript.
gene expression
The process by which DNA information is transformed into functional molecules
transcription bubble
The site at which the DNA double helix is unwound (~17 bases) so that RNA polymerase can use one of the DNA strands as a template for RNA synthesis. Elongation occurs AT this site. Consists of the RNA polymerase, DNA, and the RNA product. The structure moves along the DNA as DNA is unwound and then rewound, and the RNA product exits the enzyme and the transcribed DNA rejoins its partner.
1) Formation of an aminoacyl adenylate from an amino acid and ATP. 2) Transfer of the aminoacyl group of the aminoacyl-AMP to a particular tRNA molecule to form aminoacyl-tRNA. The ΔG∘′ of this reaction is close to 0 because the free energy of hydrolysis of the ester bond of the aminoacyl-tRNA is like that for the hydrolysis of ATP to AMP and PPi.
What are the 2 steps of the activation of amino acids by attachment to a transfer RNA by aminoacyl-tRNA synthetases?
1) Nucleotides are modified 2) Small nucleolar ribonucleoproteins methylate specific nucleoside groups and convert selected uridines into pseudouridines. This is catalyzed by RNA-protein complexes called snoRNP (small nucleolar ribonucleoproteins) in small subunits of processomes. 3) The pre-rRNA is cleaved and packaged to form mature ribosomes.
What are the 3 steps to the processing of eukaryotic pre-rRNA?
1) calcium phosphate DNA precipitation (low efficiency, but easy) 2) DNA microinjection (better success, but more difficult) 3) Retroviral injection of DNA (most effective; Moloney murine leukemia virus and Vaccina virus in mammalian cells; Baculovirus in insect cells) 4) Introduction of soil bacterium, Agrobacterium tumefacians
What are the 4 common ways to express eukaryotic genes in eukaryotes?
mRNA < tRNA < rRNA
What are the three most common types of RNA, in order of relative amount?
A. 7-methylguanosine on the 5' end of RNA
What is a 5' cap, structurally? A. 7-methylguanosine on the 5' end of RNA B. A sequence of adenosine residues added to the 5' end of the RNA C. 3-methyluracil added to the 5' end of the RNA D. 5-methylguanosine on the 3' end of the RNA E. Methylation of the 5' base of RNA, preventing further addition of bases
mRNA
What is the only RNA that gets transcribed AND translated?
TFI
What is the transcription factor for RNA polymerase I?
TFIII
What is the transcription factor for RNA polymerase III?
transfer RNA
What molecule contains the following characteristics?: 1) each single strand of RNA between 73 and 93 ribonucleotides in length 2) 3-D structure of the molecule is L-shaped 3) In 2D representation, all tRNA molecules appear as a cloverleaf pattern 4) The amino-accepting region is the acceptor stem, which contains the 3'CCA terminal region. 5) Many of the nucleotides are involved in hydrogen bonds that form stems and loops, with the anticodon being in a loop near the center of the sequence. 6) The 5' end is phosphorylated and the 5' terminal residue is usually pG 7) tRNA molecules contain unusual bases such as inosine or bases that have been modified 8) the anticodon is available to interact with an appropriate codon on mRNA while the end that is linked to an activated amino acid is well-positioned to participate in peptide-bond formation.
2a, 1b, 1b', and 1 omega
What subunits make up the core enzyme that is required for polymerization activity?
B. It is a sequence in the promoter region of genes that marks transcription start sites.
Which of the following best describes the TATA box in DNA? A. It is a sequence in chromosomes that marks replication origins. B. It is a sequence in the promoter region of genes that marks transcription start sites. C. It is a sequence in terminator region of genes that marks cleavage and polyadenylation sites. D. It is a sequence in primary transcripts that marks splice sites to remove introns. E. It is a sequence in mRNAs that marks translation start sites.
A. alternative splicing
Which of the following posttranscriptional processes can lead to the production of multiple protein isoforms from a single gene? A. alternative splicing B. capping C. nuclear export D. polyadenylation E. phosphorylation of the CTD
rho-dependent termination
a form of termination in RNA synthesis in bacteria where a hexameric rho protein helps terminate transcription. The rho protein is an ATP-dependent helicase that binds to a particular segment on the nascent RNA strand and pulls it away from RNA polymerase and the DNA template. The rho protein chases down the polymerases in the transcription bubble, and contact with the rho protein causes the transcription bubble to dissociate.
Rho-independent (intrinsic) termination
a form of termination in RNA synthesis in bacteria. An RNA hairpin followed by several uracil residues terminates the transcription. The RNA complement of the DNA stop signal forms a hairpin structure, followed by several uracil residues. Upon synthesis of the hairpin, the polymerase stalls, the RNA product is released, and the DNA double helix reforms.
translocon
a multisubunit assembly of integral and peripheral membrane proteins that functions as a protein-conducting channel. This channel opens when the assembly and ribosome bind to each other. Protein synthesizes resumes with the growing polypeptide chain passing through the channel into the lumen of the ER.
signal-recognition particle (SRP)
a protein-RNA complex consisting of a 7S RNA and six other proteins (one which is SRP54, a GTPase) that recognizes the signal sequence and binds the sequence and the ribosome as soon as the signal sequence exits the ribosomes. It will sample all ribosomes until it locates one exhibiting a signal sequence. After SRP is bound to the signal sequence, interactions between the ribosome and the SRP occlude the elongation-factor-binding site, thereby halting protein synthesis.
signal sequence
a sequence of 9-12 hydrophobic residues, along with positively charged residues, which forms an alpha helix near the N-terminal end of the nascent protein and identifies it as one that must cross the ER membrane. This results in the halting of synthesis and the ribosome complex being directed to the ER.
palindromic DNA
a sequence of DNA that is the same going forward and backwards; a transcribed product of this segment is the simplest form of a stop signal in RNA in bacteria
Zinc Finger Nucleases (ZFNs)
a sequence specific nuclease that is a DNA-binding domain compromising of 3 Cys2His2 zinc-finger domains. Each domain is stabilized by bound zinc ion through interactions with cysteine residues and two histidine residues. The preferred DNA-binding sequence can be altered by this domain by changing the identity of only four contact residues within each zinc finger.
termination signal
a specific sequence of nucleotides that marks the end of a gene. The simples form is the transcribed product of a segment of palindromic DNA. There is more known about prokaryotic termination than eukaryotic termination The RNA complement of DNA stop signal forms a hairpin structure, followed by several uracil residues. Upon synthesis of the hairpin, the polymerase stalls, the RNA product is released, and the DNA double helix reforms.
sigma subunit
a subunit of bacterial RNA polymerase that helps the polymerase find a site where transcription begins, participates in the initiation of RNA synthesis, and then dissociates from the rest of the enzyme. It can be considered the apoenzyme of RNA polymerase. It decreases the affinity of RNA polymerase for general regions of DNA and thus specifically enables the RNA polymerase to recognize promoter sites. After binding, it can quickly dissociate from the rest of the enzyme and find another enzyme to attach and help. Thus, one subunit can aid many polymerases.
RNA polymerase I
a type of RNA polymerase located in the nucleolus and is in specialized nucleoli, where it transcribes the tandem array of genes for 18S, 5.8S, and 28S in rRNA. This RNA polymerase is notably insensitive to alpha-Amanitin toxins.
RNA polymerase III
a type of RNA polymerase located in the nucleoplasm that transcribes tRNA and 5S rRNA. It responds to promoters WITHIN THE GENES to be transcribed. It is moderately sensitive to alpha-Amanitin (this polymerase is inhibited in high concentrations)
cytoplasm
all eukaryotic proteins start their synthesis in the ___________ of the cell.
anticodons
aminoacyl-tRNA synthetases recognize their tRNA partners primarily based on their ________________.
actinomycin
an antibiotic that binds in between the bases of a double-stranded DNA, preventing it to be used as a template, and inhibiting both prokaryotic and eukaryotic transcription.
rifampicin
an antibiotic that inhibits bacterial transcription initiation by binding to a pocket in the channel that is normally occupied by the newly formed RNA-DNA hybrid. By doing this, the antibiotic blocks elongation after only 2 or 3 nucleotides have been added.
genome editing
an experimental method of detecting and correcting defective genes in an individual; relies on the introduction of double-stranded breaks at precisely determined sequences within genomic DNA; The resulting cleavage site is repaired by the DNA repair machinery of the host cell in one of the two ways: 1) If no template, the cell will repair using non-homologous end joining (NHEJ), which is error-prone and will introduce a lot of insertions or deletions into the repair site. 2) If DNA fragment containing desired sequence change is simultaneously introduced with a nuclease, the repair machinery will use donor template to introduce the changes directly into the genomic sequence; aka homology directed repair (HDR)
CTD
carboxyl-terminal domain, which is a unique structure found in RNA polymerase II. It contains multiple repeats of a YSPTSPS consensus sequence and plays an important role in regulation. Phosphorylation on the serine residues in this domain is important for RNA polymerase II regulation. -Functions: 1) Recruiting enzymes to synthesize the 5' cap in eukaryotic mRNA 2) Recruiting components of the splicing complex in eukaryotic mRNA 3) Recruiting the endonuclease that cleaves the pre-mRNA in eukaryotes to expose the 3' end site for a poly A tail addition
ribozymes
catalytic RNA molecules that function as enzymes and can splice RNA. Can catalyze two types of reactions: RNA cleavage and RNA transesterification reactions. Their substrates are positioned in the active site and normally use a guide sequence that is complementary to the substrate. A key note is that they all depend absolutely on the assumption of correct 3D structures for activity. Ex) RNase P, group I and II introns, ribosomes, and hammerheads.
genetic code
collection of codons of mRNA, each of which directs the incorporation of a particular amino acid into a protein during protein synthesis. Characteristics: 1) codon (3 nucleotides) encode an amino acid 2) The code is non-overlapping 3) The code has no punctuation 4) Code has directionality (5' to 3' end) 5) Code is degenerated in that some amino acids are encoded by more than one codon (this minimizes the deleterious effects of mutations) 6) The code is nearly universal
anticodon
group of three bases on a tRNA molecule that are complementary to an mRNA codon
The RNA world hypothesis
hypothesis that RNA served as the genetic information of early life; Occurs in 5 steps: 1) RNA forms from inorganic sources 2) RNA self-replicates via ribozymes 3) RNA catalyzes protein synthesis 4) Membrane formation changes internal chemistry, allowing new functionality 5) RNA codes both DNA and protein RNA --> RNA --> protein
the nuclear membrane
in eukaryotes, transcription and translation takes place in different cellular compartments within the bounds of this structure (transcription takes place in the membrane-bound nucleus, whereas translation takes place outside the nucleus in the cytoplasm). The spatial and temporal separation of transcription and translation enables eukaryotes to regulate gene expression in much more intricate ways, contributing to the richness of eukaryotic form and function.
5' to 3' amino-to-carboxyl
in protein synthesis (translation), mRNA is decoded in what direction, one codon at a time? The corresponding protein is synthesized in what direction by the sequential addition of amino acids?
RNA interference (RNAi)
introduction of double-stranded RNA into a cell to inhibit gene expression (but does not eliminate it). Occurs in 3 stages: 1) The introduced double-stranded RNA is cleaved into small fragments by the enzyme dicer to produce small interfering RNA (siRNA) 2) The fragment that is complementary to the mRNA becomes incorporated into the RNA-induced silencing complex (RISC) 3) The associated complementary RNA guides the complex to the mRNA, which is then degraded.
proteins
linear polymers of amino acids that spontaneously fold up into 3D structures.
uracil, thymine
mRNA and coding strand DNA possess the exact same sequence, except mRNA contains the base ________, while the coding strand contains the base _______ in place of mRNA's base.
exon shuffling
mechanism of when exons are mixed up by the DNA recombination to expand the genetic repertoire. This process generated the tissue plasminogen activator (TPA)
mRNA
messenger RNA; type of RNA that carries instructions from DNA in the nucleus to the ribosome/encodes the information to generate a protein; acts as a translation (protein synthesis) regulatory
elongation mechanism
nucleotide addition; A ribonucleoside triphosphate binds adjacent to the growing RNA chain and forms a Watson-Crick base pair with a base on the DNA template strand. The 3´-hydroxyl group at the end of the RNA chain attacks the newly bound nucleotide and forms a new phosphodiester bond, releasing pyrophosphate.
cassette mutagenesis
one of the methods for introducing mutations into DNA; allows more complex sets of mutations. An oligonucleotide (aka a cassette) containing the mutations is interested into the gene of interest.
site-direct mutagenesis
one of the methods for introducing mutations into DNA; allows the generation of mutant proteins with a single amino acid substitution.
mutagenesis by PCR
one of the methods for introducing mutations into DNA; uses primers to introduce insertions, deletions, and substitutions.
transgenic organisms
organism that contains genes from other organisms; are important tools for investigating a variety of diseases. Ex) Versions of mice to better understand ALS.
RNA editing
process of altering the nucleotide sequence of an mRNA post-transcriptionally without RNA splicing. -A base in the mRNA is changed to alter the information content of the mRNA
rRNA
ribosomal RNA; type of RNA that makes up part of the ribosome; active in translation (protein synthesis) and is catalytic
scRNA
small cytoplasmic RNA; acts as a signal recognition particle (SRP) and in tRNA processing; is catalytic and includes RNase P
snRNA
small nuclear RNA; acts in processing of pre-mRNA and poly A addition in the cytoplasm and eukaryotes; is catalytic
snoRNA
small nucleolar RNA; processing/maturation/methylation/assembly of RNA
promoters
specific regions of DNA sequences that direct RNA polymerase to the proper initiation site
transcription
synthesis of an RNA molecule from a DNA template; driven thermodynamically by the hydrolysis of pyrophosphate; The process begins near promoter sites and ends at terminator sites
holoenzyme
the addition of a sigma factor to the core enzyme creates the _____________, which is required for correct initiation of transcription (binding to promoter) so that the core enzyme can activate elongation.
protein targeting and sorting
the delivery of newly formed ribosomes by eukaryotic cells to internal sites such as the mitochondria, the nucleus, and the ER. This process is accomplished by two general mechanisms. 1) Proteins destined for nucleus, chloroplasts, mitochondria, and peroxisomes: The protein is synthesized in the cytoplasm, and then the completed protein is delivered to its intracellular location post-translationally. 2) "The secretory pathway:" directs proteins into the ER (extensive membrane system that compromises about half the total membrane of a cell), co-translationally. Approx. 30% of all proteins are sorted through this mechanism included secreted proteins, residents of the ER, the golgi complex, lysosomes, and integral membrane proteins of these organelles as well as integral plasms-membrane proteins.
homologous recombination
the exchange of genetic information (via segments) of two similar DNA molecules; leads to genes being disrupted or knocked out. While it is a powerful tool, there are limitations. Recombination at the desired site can be inefficient and time-consuming. The method also is generally limited to specific model organisms such as yeast and mice.
RNA polymerase II
the wide range of promoters such as the TATA box, initiator element (Inr), downstream promoter element (DPE), and/or TBGFIIB recognition element (BRE) control which RNA polymerase?
central dogma
theory that states that, in cells, information only flows from: DNA -> RNA -> Protein
DNA microarrays
tools that allow scientists to study many genes, and their expression, at once. Allows for the determination of the expression patterns of many genes simultaneously. DNA sequences are attached to a solid support in a defined pattern to generate the microarray. Fluorescently labeled cDNA is then hybridized to the microarray to reveal the expression level of each gene on the chip.
tRNA
transfer RNA; type of RNA that acts as a specific adaptor RNA that carries amino acids to ribosomes and mRNA, and connects codes between mRNA and the protein that is going to be synthesized. It is active in translation (protein synthesis). They react with specific amino acids in a reaction catalyzed by aminoacyl-tRNA synthetases. Contain anticodons that consists of 3 bases that recognize a complementary 3-base sequence in mRNA.
ribosomal RNA
type of RNA that combines with proteins to form ribosomes; is considered the actual catalyst for protein synthesis.
Quantitative PCR (qPCR)
variation that quantifies initial amount of DNA; it is used to determine how much of a particular gene is expressed compared to another gene. It measures how much individual mRNA is expressed compared to a housekeeping gene. The PCR cycle at which fluorescence's become detectable is inversely related to the original number of target templates. In one approach, the transcript of interest is PCR amplified with the appropriate primers in the presence of dye SYBR green, which fluoresces brightly when bound to double-stranded DNA. After repeated PCR cycles, fluorescence intensity exceeds detection threshold and continues to rise as the number of duplexes corresponding to the transcript of interest increases (the number is inversely proportional to the number of copies of the original template). After relation between the original copy number and the CT (threshold) have been established with the use of a known standard, this experiment can be used to determine the number of copies of any desire transcript in the original sample.
housekeeping and myosin
what 2 cell types are in muscle cells?
housekeeping and insulin
what 2 cell types are in pancreatic cells?
housekeeping and hemoglobin
what 2 cell types are in red blood cells?
All RNA
what RNA is transcribed?
rifampicin and actinomycin
what are 2 antibiotics that inhibit transcription?
the nuclear membrane, mRNA processing, and more complex transcriptional regulation.
what are 3 important characteristics that influence gene expression in eukaryotes?
RNA molecules
what are central in the central dogma?
rho-independent (intrinsic) and rho-dependent
what are the 2 main types of termination of RNA synthesis in bacteria?
rRNA, scRNA, and snRNA
what are the 3 catalytic types of RNA?
1) acquisition of 5'-methylguanylate cap 2) splicing of introns 3) acquisition of a poly A tail on the 3' end *They are coordinated by the phosphorylated CTD of RNA polymerase II
what are the 3 factors of transcription and processing in mRNA of eukaryotes?
deletions, insertions, and substitutions
what are the 3 main classes of mutations?
1) spliceosome assembly and activation 2) splicing catalysis 3) snRNP recycling
what are the 3 stages of spliceosome action?
1) OligoT (thymine) residues are used with the poly (A) sequence at the 3' end of the most eukaryotic mRNA molecules. The reverse transcriptase then synthesizes the rest of the cDNA strand in the presence of he four deoxyribonucleoside triphosphates. 2) The template RNA strand of this RNA-DNA hybrid is subsequently hydrolyzed by raising the pH. 3) Unlike RNA, DNA is resistant to alkaline hydrolysis. The single-stranded DNA is converted into double-stranded DNA by creating another primer site. The enzyme terminal transferase adds nucleotides to the 3' end of DNA. 4) Oligo(dC) can bind to dG residues and prime the synthesis of the second DNA strand
what are the 4 steps of cDNA synthesis?
alpha, beta, beta prime, and omega
what are the core subunits in RNA polymerase in e.coli?
10^-4
what is considered the most "efficient" error frequency that allows for the accurate synthesis of even large proteins at a very rapid rate?
DNA --> Transcription --> Pre-mRNA --> Processing --> mRNA --> Translation --> Protein --> Maturation --> Folding/localization subunit assembly --> Biological function --> Degradation
what is the "life cycle" of a protein?
1) Amino acids are carried into the ribosome and connected to transfer RNA molecules. 2) Each amino acid is then joined to the growing polypeptide chain within the ribosome, which detaches from the ribosome only after the polypeptide has been completed. This assembly-line approach allows even very long polypeptide chains to be assembled rapidly and with impressive accuracy.
what is the general theme/concept of protein synthesis?
eukaryotes separate transcription and translation with a nucleus
what is the most significant influence on why mRNA is processed differently in prokaryotes than eukaryotes?
TFII -TFIIA, TFIIB, etc.
what is the transcription factor for RNA polymerase II?
bacteria RNA polymerase
what synthesized all RNA in bacteria (occurs in 3 stages: initiation, elongation, termination)?
-10 and -35 sequences
what two common promoters make up the core promoter in e.coli?
nucleic acids and protein factors
what two critical parts does protein synthesis depend on?
ribosomal initiator (rlnr) and an upstream promotor element (UPE)
what two factors does RNA polymerase I promoter consist of?
