Bio Test #2

attachment of amino acids to specific tRNAs is mediated by aminoacyl tRNA synthetases

20 enzymes that recognize a single amino acid and the correct tRNAs amino acid is activated by reaction with ATP to form an aminoacyl AMP activated amino acid is joined to 3' terminus of tRNA highly selective enzymes that recognize the amino acid and base sequence that identify the correct acceptor tRNAs

formation of 3' ends of eukaryotic mRNAs

3' end is defined by cleavage of the primary transcript and addition of a poly-A tail (polyadenylation) -signals include a highly conserved hexanucleotides (10 - 30 nucleotides upstream of the site of polyadenylation) and a U- or GU-rich downstream sequence element -sequences recognized by a complex of protein, including an endonuclease that cleaves the RNA chain and a poly-A polymerase that adds a poly-A tail to the transcript -recognition of the polyadenylation signal leads to termination of transcription, cleavage and polyadenylation of the mRNA, followed by degradation of the RNA that has been synthesized downstream -these processing enzymes are associated with the phosphorylated CTD of RNA polymerase II poly-A tails regulate translation and mRNA stability -polyadenylation plays important role in early development, where changed in length of poly-A tails control mRNA translation

chromosome conformation capture (3C)

3C analysis has indicated that genomes are divided into a series of looped domains, such that regions within a domain interact frequently with one another, but rarely interact with region of other domains 3C -cross-linked adjacent DNA --> PCR -if they got amplification of DNA --> pieces are close to each other

in eukaryotic cells, the ubiquitin-proteasome pathway is the major mediator of regulated protein degradation

76 - amino acid polypeptide that is attached to the amino groups of the side chains of lysine residues Ubiquitin is activated by being attached to an ubiquitin-activating enzyme, E1 -ubiquitin is transferred to a second enzyme, called ubiquitin-conjugating enzyme (E2) -ubiquitin is transferred to the target protein by E2 complexed with a third protein, called ubiquitin ligase (E3) -E3s mediate the selective recognition of target proteins by binding to both a substrate and an E2 proteins targeted for degradation are marked by the addition of multiple ubiquitins to form a polyubiquitin chain, which is catalyzed by some E3s -polyubiquinated proteins are recognized and degraded by proteasome

hereditary breast cancer

BRCA2 is a gene responsible for breast cancer -encodes a protein that acts to recruit Rad51 to the single-stranded DNA generated at double-strand breaks -defects in repair of double-stranded DNA can lead to development of breast cancer

A eukaryotic promoter

Cis-acting sequences serve as binding sites for a variety of gene-specific regulatory factors that control the expression of individual genes -required for efficient transcription -binding for gene-specific TFs -located upstream of transcription start site -two regulatory sequences found in many eukaryotic genes were identified in the promoter of the herpes simplex virus

activators and repressors regulate transcription by inducing changes in the structure of chromatin

DNA of all eukaryotic cells is tightly bound to histones histones modification is a key mechanism for regulating the expression of eukaryotic genes modifications of histones are stably inherited when cells divide, so they provide a key mechanism through which patterns of gene expression can be transmitted to progeny

replication factories

DNA replication takes place within discrete regions called replication factories -contain clustered sites of DNA replication -clustered sites of newly synthesized DNA represent concentrated sites of the proteins involved in DNA replication (PCNA), indicating that they are large complexes of proteins engaged in DNA synthesis

promoter

DNA sequence to which RNA polymerase binds to initiate transcription of a gene region upstream of transcription initiation site contains two sets of sequences that are similar in a variety of gene - -10 and -35 elements -σ subunit bind specifically to sequences in both the -35 and -10 promoter region σ subunit directs the polymerase to promoters by promoter binding specifically to both the -35 and -10 sequences, leading to initiation of transcription at the beginning of the gene -polymerase unwinds to form an open-promoter complex in which single-stranded DNA is available as a template for transcription -transcription is initiated by joining of two free NTPs -after the first 10 nucleotides, σ subunit is released from polymerase -polymerase leaves the promoter and moves along the template DNA to continue elongation of the RNA chain

identification of eukaryotic regulatory sequences

Eukaryotic regulatory sequences are added upstream of the reported gene that encodes an easily detectable enzyme (firefly luciferase) expression of reporter gene following its transfer into cultured cells provides a sensitive assay for the ability of the cloned regulatory sequence to direct transcription active regulatory regions can be identifies, and in vitro mutagenesis can be used to determine the role of specific sequences within the region

chaperones stabilize unfolded polypeptide chains during their transport into subcellular organelles

Ex: transfer of proteins into mitochondria from cytosol -proteins are transported across the mitochondrial membrane in partially unfolded conformations that are stabilized by chaperones in the cytosol -chaperones within the mitochondrion facilitate transfer of the polypeptide chain across the membrane and its subsequent folding within the organelle

Many genes in mammalian cells are controlled by regulatory sequences located far away from transcription start site

Known as enhancers SV40 enhancer -in addition to a TATA box and a set of 6 GC boxes, two 72-base-pair repeats located farther upstream are required for efficient transcription from this promoter could stimulate transcription no matter where placed

Three types of lipid additions are common in eukaryotic proteins associated with the cytosolic face of the plasma membrane

N-myristoylation, prenylation, and palmitoylation -attachment of fatty acid to amino terminus of protein N-myristoylation: initiating methionine is removed, leaving glycine at the N-terminus of the polypeptide chain -myristic acid is added (myristolation) -many plasma membrane-associated proteins involved in the control of cell growth and differentiation are modified in this way

protein are targeted for export from nucleus by specific amino acid sequences called nuclear export signals (NES)

NES are recognized by receptors within the nucleus (exportins), which direct protein transport through the nuclear pore complex to cytoplasm exportins bind to Ran -Ran/GTP promoted the formation of stable complexes between exportins and their cargo proteins -this effect of Ran/GTP binding to exportins dictates the movement of proteins containing nuclear export signals from the nucleus to the cytoplasm -following transport to the cytosolic side of the nuclear envelope, GTP hydrolysis and release of Ran/GDP leads to dissociation of the cargo protein, which is released into the cytoplasm -exportins and Ran/GDP are recycled through the nuclear pore complex for reuse

VDJ recombination is mediated by the two proteins RAG1 and RAG2, which are expressed in lymphocytes

RAG proteins recognize recombination signal (RS) sequences adjacent to the coding sequence of each gene segment, and initiate DNA rearrangements by introducing a double-strand break between the RS sequences and the coding sequences -cleave DNA -coding ends of the gene segments are joined to yield a T receptor gene -joining reaction is frequently accompanied by loss of nucleotides -lymphocytes contain an enzyme (terminal deoxynucleotidyl transferase) that adds nucleotides at random at the ends of DNA molecules -these mutations contribute to the diversity of immunoglobulins and T cell receptors

splicing takes place in large complexes (spliceosomes) composed of proteins and RNAs

RNA components are 5 small nuclear RNAs (snRNAs) called U1, U2, U4, U5, and U6 -complexed with 6 to 10 protein molecules to form small nuclear ribonucleoprotein particles (snRNPs), which play central roles in splicing process U1, U2, and U5 snRNPs contain a single snRNA molecule U4 and U6 snRNAs are complexed to each other in a single snRNP first step in spliceosome assembly is binding of U1 snRNP to 5' splice site of pre-mRNA by base pairing between the 5' splice site consensus sequence and a complementary sequence at the 5' end of U1 snRNA -U2 snRNP binds to the branch point -preformed complex of U4/U6 and U5 snRNPs is incorporated into the spliceosome -U4 and U1 dissociate from the spliceosome -splicing reaction is accompanied by rearrangements of the snRNAs, with the reaction catalyzed by U2, U5, and U6 to form a loop of introns and ligation of exons

Transcription of RNA polymerase I

RNA polymerase I is devoted solely to transcription of ribosomal RNA genes -cells contain multiple tandem copies of genes encoding the 5.8S, 18S, and 28S rRNAs, which transcripts as a unit within the nucleolus -transcription yields a 45S pre-rRNA, which is cleaved to yield the 28S, 18S, and 5.8S rRNAs -promoter of rRNA genes are at -150 ~recognized by two transcription factors: UBF and SL1, which bind cooperatively to the promoter and recruit polymerase I to form an initiation complex

RNA editing

RNA processing events that alter the protein-coding sequences of some mRNAs editing in mammalian nuclear mRNAs involved single base changes RNA editing reactions include the deamination of cytosine to uridine ex: editing of mRNA for for apolipoprotein B, which transports lipids in the blood -tissue-specific RNA editing results in two different forms of apolipoprotein B -in humans, Apo-B100 is synthesized in liver by translation of the unedited mRNA -shorter protein Apo-B4B8 is synthesized in the intestine as a result of translation of an edited mRNA in which a C is changed to a U by deamination -alteration changes codon for glutamine to translation for stop codon --> shorter Apo-B protein -results in structurally different protein in liver and intestine

regulation of transcriptional elongation

Transcription by RNA polymerase II initiates following phosphorylation of CTD serine 5 by the TFIIH protein kinase -polymerase synthesizes a short region of RNA and then pauses near the beginning of the gene -arrest of the polymerase results from the association of a negative regulatory factor such as NELF and DSIF that prevent further transcription continuation depends on P-TEFb -contain a protein kinase that phosphorylates NELF and DSIF as well CTD serine 2 -leads to initiation of productive elongation and association of additional elongation and processing factors with the CTD

Action of enhancers

Without an enhancer, the gene is transcribed at a low basal level addition of an enhancer stimulates transcription when -placed just upstream the promoter -inserted up to several kb upstream or downstream transcription start site -either forward or backward orientation

protein folding

all the information required for a protein to adopt the correct 3-D conformation is provided by its amino acid sequence denatured RNase can spontaneously refold in vitro to its active conformation -this occurs too slowly to be useful within a cell disulfide bonds

Translation initiation

always initiated by methionine, usually encoded by AUG -in bacteria, protein synthesis is initiated with a modified methionine residue -in eukaryotes, unmodified methionine initiates protein synthesis initiation codons in bacterial mRNAs are preceded by Shine-Dalgarno sequence, which aligns the mRNA on the ribosome by base-pairing with complementary sequence near the 3' terminus of 16S rRNA -this allows bacterial ribosomes to initiate translation at 5' end of mRNA and at the internal initiation sites of polycistronic messages ribosomes recognize eukaryotic mRNAs by binding to the 7-methyl-guanosine cap at 5' terminus -ribosome scans downstream of 5' cap until it encounters the initiation codon several viral and cellular mRNAs have internal ribosome entry sites (IRES) at which translation can initiate by ribosome binding to an internal position on the mRNA -function under conditions of cell stress to allow selective translation of some mRNAs when normal mode of initiation at 5' cap is inhibited

Mediator is required to stimulate transcription

associated with RNA polymerase II and general transcription factors stimulates basal transcription and plays a key role in linking the general transcription factors to the gene-specific transcription factors that regulate gene expression transcription initiates following phosphorylation of the polymerase CTD by the TFIIH protein kinase -releases polymerases from Mediator and other transcription factors -CTD binds additional proteins that facilitate transcriptional elongation

histone modification

basic structural unit of chromatin is the nucleosome -147 base pairs of DNA wrapped around two molecules of each histone structure of chromatin can be altered by modifications of histones and by rearrangements of nucleosomes

transcriptional activators

bind to regulatory DNA sequences and stimulate transcription -have 2 domains: one region binds DNA, and the other stimulates transcription by interacting with other proteins -DNA binding domain anchors the transcription factor to the proper site on DNA -activation domain independently stimulates transcription through two distinct mechanisms -activation domains interact with Mediator proteins and general transcription factors (TFIIB or TFIID) to recruit RNA polymerase and facilitate assembly of transcription complex on promoter -activation domains interact with coactivators that facilitate transcription by modifying chromatin structure.

translation regulation

binding of repressor proteins (block translation) to specific mRNA sequences Ex: regulation of ferritin, a protein that stores iron in the cell -translation of ferritin mRNA is regulated by supply of iron: more ferritin is synthesized if iron is abundant -mediated by iron regulatory proteins (IRPs) that, in the absence of iron, bind to the iron response element (IRE) in the 5' untranslated region of ferritin mRNA, blocking translation -IRPs repress translation by interfering with binding of 40S ribosomal subunits to mRNA

regulation of protein function

binding of small molecules -changes shape and function -ex: lac repressor (allosteric regulation) phosphorylation protein-protein interactions

regulation of eIF4E

binds to 5' cap of mRNA -critical point at which growth factors act to control protein synthesis ex: growth factors that stimulate protein kinases that phosphorylate regulatory protein (4E-BPs) that bind to eIF4E -in absence of appropriate growth factors, nonphosphorylated 4e-BPs bind to eIF4E and inhibit translation by interfering with the interaction between eIF4E and eIF4G -growth factor stimulation leads to the phosphorylation of 4E - BPs, which dissociates from eIF4E, allowing translation to proceed mRNAs containing internal ribosome entry sites (IRES) may be selectively translated under conditions that inhibit eIF4E and thereby lead to inhibition of cap-dependent initiation -help cell cope with cellular stress

Some RNAs are capable of self-splicing

catalyze removal of their own introns first described during studies of 28S rRNA of the protozoan Tetrahymena -RNA has an intron that is precisely removed following incubation of pre-rRNA in absence of added protein splicing is catalyzed by the intron (act as a ribozyme to direct its own excision) first step in splicing for group I introns is cleavage at 5' splice site mediated by a guanosine (G) cofactor -3' end of the free exon then reacts with the 3' splice site to excise the intron as a linear RNA active catalytic component of spliceosomes are RNAs rather than proteins -pre-mRNA splicing was catalyzed by the snRNAs of the spliceosome -protein components of snRNPs are also required for assembly of the spliceosome and the anchor the snRNAs during splicing

protein phosphorylation

catalyzed by protein kinases, most of which transfer phosphate groups from ATP to the hydroxyl groups of the side chains of serine, threonine, or tyrosine residues -these enzymes are called serine/threonine kinases or tyrosine kinases, which catalyze the hydrolysis of phosphorylated amino acid residues -protein phosphorylation is reversed by protein phosphatases protein kinases function as components of signal transduction pathways in which on kinase activates a second kinase, which may act on another --> react to environmental stimuli

glycoproteins

classified as either N-linked or O-linked, depending on site of attachment of the carbohydrate side chain -in N-linked glycoproteins, the carb is attached to the nitrogen atom in the side chain of asparagine -in O-linked glycoproteins, the oxygen atom in the side chain or serine or threonine is the site of carbohydrate attachment -the sugars directly attached to these positions are either N-acetylglucosamine or N-acetylgalactosamine most glycoproteins in eukaryotic cells are destined for secretion or for incorporation into plasma membrane -these proteins are usually transferred into the ER while their translation is still in progress and glycosylation occurs in the ER and Golgi -cytoplasmic and nuclear proteins are modified by the addition of single O-linked N-acetylglucosamine residues in the cytosol

protein cleavage

cleavage of polypeptide chain (proteolysis) is an important step in the maturation of many proteins active enzymes or hormones form via cleavage of larger precursors - ex: insulin -forms by two cleavages -initial precursor (preproinsulin) contains an amino-terminal signal sequence that targets the polypeptide chain to the ER -removal of signal sequence during transfer to ER yields a second precursor called proinsulin -proinsulin is converted to insulin by proteolytic removal of connecting polypeptide other proteins activated by similar cleavage processes including digestive enzymes, proteins invovled in blood clotting, and a cascade of proteases that regulate programmed cell death

The nuclear envelope

complex structure consisting of two nuclear membranes, an underlying nuclear lamina, and nuclear pore complexes nucleus is surrounded by two concentric membranes, called the inner and outer nuclear membranes -outer nuclear membrane is connected with the lumen of the ER ~functionally similar to the membranes of the ER and has ribosomes, but differs in protein composition -inner membrane contains 60 specific integral membrane proteins, like those that bind the nuclear lamina nuclear membrane separated the contents of the nucleus from the cytoplasm -each nuclear membrane is a phospholipid bilayer permeable only to small nonpolar molecules inner and outer nuclear membranes are joined at nuclear pore complexes (sole channels through which small polar molecules and macromolecules pass through the nuclear envelope) -responsible for selective traffic of proteins and RNAs between the nucleus and cytoplasm nuclear lamina: underlying the inner nuclear membrane -filamentous meshwork that provides structural support to the nucleus -composed of 60 to 80 lamins, along with associated proteins -class of intermediate filament proteins

model of nuclear pore complex

consists of an assembly of 8 spokes arranges around a central channel -spokes connected to rings at nuclear and cytoplasmic surfaces -spoke-ring assembly is anchored within the nuclear envelope at sites of fusion between the inner and outer nuclear membrane -protein filaments extend from cytoplasmic and nuclear rings, forming a basket-like structure on the nuclear side

histone acetylation

correlated with transcriptionally active chromatin in a wide variety of cell types core histones have an amino-terminal tail (extends outside nucleosome), and a histone-fold (involved in interactions with other histones and in wrapping DNA around the nucleosome core particle) -amino-terminal tails are rich in lysine and can be modified by acetylation (adding acetyl group) of side chain of specific lysine residues, which neutralizes the positive charge of lysine and appears to contribute to transcriptional activation by relaxing chromatin structure and increasing availability of DNA template to transcription factors and RNA polymerase -H3K14ac = activation

double-strand breaks

dangerous because the continuity of the DNA molecule is disrupted by breaks in both strands occur naturally during DNA replication from a nick in template strand or ionizing radiation and chemicals that introduce breaks in opposite strands repaired by recombinational repair, which rejoins the broken strands

Translesion Repair

deal with damaged DNA at replication fork but not normal bases -no longer have undamaged opposite strand for excision repair provide a mechanism by which the cell can bypass DNA damage at the replication repair In E.coli, the first specialized DNA polymerase is DNA polymerase V (induced in response to UV and can synthesize a new DNA strand across from a thymine dimer) -also polymerases V and IV -specialized DNA polymerases replace a normal polymerase that is stalled at a site of DNA damage -synthesize across site of damage, and are replaced by the normal replicative polymerase and nucleotide excision repair occurs -exhibit low fidelity when copying undamaged DNA, and lack 3' to 5' proofreading --> high error rate -display some selectivity in inserting the correct base opposite certain lesions (thymine dimers) -error prone in inserting bases opposite other forms of damaged DNA or in the synthesis of DNA from a normal undamaged template

Types of DNA damage

deamination of cytosine occurs spontaneously in DNA UV light induces formation of pyrimidine dimers in which two adjacent pyrimidines (thymine) are joined by a cyclobutane ring structure carcinogens react with DNA bases, resulting in addition of large bulky chemical groups to the DNA molecule

chromatin immunoprecipitation

determine the regions of DNA that bind a transcription factor within the living cell -cells are treated with formaldehyde, which cross-links chromatin and transcription factors to DNA -chromatin is extracted and sheared to 500 base pairs -fragments of DNA linked to a transcription factor of interest can be isolated by immunoprecipitation with an antibody against the transcription factor -collect chromatin-antibody complex -cross-links reversed, and immunoprecipitated DNA is isolated and analyzed binding of a transcription factor to a specific regulatory element is tested using PCR to detect the sequence of interest in chromatin -can use genome-wide analysis of the immunoprecipitated DNA fragments by large-scale DNA sequencing or by hybridization to microarrays --> identify all the sites to which transcription factor is bound (ChIP)

chromatin domains that are localized to the periphery of the nucleus have been identified by use of chromatin immunoprecipitation to isolate regions of DNA that are associated with nuclear lamins

domains that interact with nuclear lamina are called lamina-associated domains (LADs) -genes found in LADs are generally transcriptionally repressed and correspond to transcriptionally inactive heterochromatin domains interactions that mediate association of heterochromatin with the nuclear lamina include binding of the lamin B receptor to heterochromatin protein 1 (HP1) -HP1 binds to methylated histone H3 lysine 9 residues (H3K9), which are characteristic of transcriptionally inactive chromatin -Also HDAC transcriptionally active domains are generally found together -domains of transcriptionally active chromatin are generally localized to either the nuclear lamina or nucleolus

regeneration of eEF1α/GTP

eEF1α needs to be reconverted to it GTP form -requires eEF1βγ, which binds to the eEF1α/GTP complex and promotes exchange of GDP for GTP -results in regeneration of eEF1α/GTP, which is ready to escort a new aminoacyl tRNA to A sire

translational regulation in eukaryotic cells that result in global effects on overall translational activity involve modulation of the activity of initiation factors (eIF2 and eIF4E)

eIF2 binds to initiator methionyl tRNA, bringing it to the ribosome -release of eIF2 is accompanied by GTP hydrolysis -eIF2/GTP complex must be regenerated by exchange of GDP for GTP, mediated by eIF2B -regulation of eIF2 provides a critical control point in eukaryotic cells -eIF2 and eIF2B can be phosphorylated by regulatory protein kinases, which inhibit exchange of GDP for GTP, inhibiting translation (happens under stress)

DNA repair: direct reversal of the chemical reaction responsible for DNA damage

efficient way of dealing with specific types of DNA damage that occur frequently -most limited because has to recognize specific kind of damage -not common Only few types of DNA damage are repaired this way -pyrimidine dimers (adjacent pyrimidines on the same strand of DNA are joined by formation of a cyclobutane ring, resulting from saturation of the double bonds between carbon 5 and 6) resulting from exposure to UV light -dimers distort the structure of the DNA chain and blocks transcription or replication photoreactivation: direct reversal of dimerization reaction -energy derived from visible light is used to break cyclobutane ring structure -common to a variety of prokaryotic and eukaryotic cells -not universal (not in humans)

termination of translation

elongation continues until a stop codon is translocated into A site -cells have release factors (RFs) that recognize the signals and terminate protein synthesis -release factors bind to a termination codon at A site and stimulate hydrolysis of bond between tRNA and polypeptide chain at P site, releasing completed polypeptide from ribosome -tRNA is released, and ribosomal subunits and mRNA dissociate

immunoglobulin enhancer

enhancers contain multiple sequence elements that bind to different transcriptional regulatory proteins -proteins work together to regulate gene expression -immunoglobulin heavy chain enhancer spans 200 bases and contains 9 protein binding sites which stimulate transcription of B lymphocytes reflects the combined action of the proteins associated with each of its individual sequence elements

glycogen breakdown is catalyzed by the enzyme glycogen phosphorylate, which is regulated by protein kinase

epinephrine binds to a cell surface receptor that triggers the conversion of ATP to cAMP, which then binds to and activates a protein kinase (cAMP-dependent protein kinase) this kinase phosphorylates and activates a second protein kinase (phosphorylase kinase) phosphorylate kinase phosphorylates and activated glycogen phosphorylate, leading to glucose production removal of initial stimulus inhibits further glycogen breakdown

mRNA export

exported by a distinct mechanism that doesn't involve karyopherins and is independent of Ran distinct mRNA exported complex is recruited to pre-mRNAs in the nucleus in concert with the completion of splicing and polyadenylation -exporter complex transports mRNAs through the nuclear pores -directionality of the process is established by an RNA helicase localized to the cytoplasmic face of the nuclear pore complex ~helicase remodels the mRNA and removes the exporter complex, releasing mRNA into cytoplasm and prevents its transport back into the nucleus

mRNA degradation

final aspect of the processing of an RNA molecule (in cytoplasm) -bacterial mRNAs are rapidly degraded, allowing cell to respond quickly to alteration in its environment in eukaryotic cells, mRNAs are degraded at different rates 5' and 3' ends of mRNAs are protected from degradation by capping and polyadenylation -degradation is initiated by shortening of poly-A tails -deadenylation mRNAs are degraded by nucleases that act from the 3' end or by removal of the 5' cap and degradation of the RNA by nucleases acting from the 5' end

DNA Repair

fix mistakes during replication damage (chemicals, spontaneous, radiations) that occur through life consequences --> mutations in codons, block replication, transcription, etc.

DNA methylation

general mechanism for epigenetic control of transcription in eukaryotes -cytosine residues in DNA of fungi, plants, and animals can be modified by addition of methyl group at 5-carbon position -DNA is methylated specifically at cytosines that precede guanines in the DNA chain (CpG islands), which correlates with transcriptional repression methylation plays key role in suppressing movement of transposons throughout the genome -associates with transcriptional repression of mammalian genes involved in development and differentiation, in concert with alterations in chromatin structure DNMT - DNA methylase

Negative control of the lac operon

genes encoding β-galactosidase, permease, and transacetylase are expressed as a single unit (operon) which have two distinct loci (o and i) that control expression of the operon transcription of the operon is controlled by o (operator), which is adjacent to the transcription initiation site i gene encodes a protein that regulated transcription by binding to the operator DNA --> repressor, which blocks transcription when bound to the o by preventing RNA polymerase from binding to the promoter addition of lactose leads to induction of the operon because allolactose binds to the repressor, preventing it from binding to the operator DNA Control of transcription is mediated by interaction of regulatory proteins with specific DNA sequences -regulatory sequences are called cis-acting control elements because they affect the expression of linked genes on the same DNA molecule -proteins like the repressor can affect the expression of genes located on other chromosomes within the cell -lac operon is negative control because binding of the repressor blocks transcription

transcription of RNA polymerase III

genes for 5S rRNA, tRNAs, and some small nuclear RNAs (snRNAs) are transcribed by polymerase III -transcribed from 3 classes of promoters -TFIIA initiates assembly of a transcription complex by binding to specific DNA sequences in the 5S rRNA promoter -followed by binding of TFIIC, TFIIB, and the polymerase -promoters for the tRNA genes and snRNAs are recognized by other factors that recruit TFIIB and the polymerase (TFIIIC and SNAP)

immunoglobulin diversity

genes that encode immunoglobulin light chains have 3 regions -V region encodes 95 to 96 N-terminal amino acids -joining (J) region encodes 12 to 14 C-terminal amino acids -C region encodes the constant region -single V region recombines with one of the 4 J regions to generate a functional light-chain gene ~random ~rearranged differently in each B lymphocyte -remaining unused J region and introns between J region and C region is spliced out, yielding a functional mRNA heavy chain contains D region (diversity) that encodes amino acids lying between V and J -D region recombines with a J region, and a V region recombines with the rearranged DJ segment joining of immunoglobulin gene segments often involve the loss or gain of one to several nucleotides, increasing diversity

Xeroderma pigmentosum (XP)

genetic disorder that affects 1 in 250,000 extremely sensitive to UV light, and develop skin cancers cultured cells from XP patients were deficient in the ability to carry out nucleotide-excision repair

heterochromatin in interphase nuclei

genome is not randomly distributed within nucleus -location of different genes varies in different cells -Heterochromatin frequently at periphery associated with lamina, actively transcribed genes at nuclear pores each chromosomes occupies a distinct territory of the nucleus (chromosome territory), with centromeres and telomeres attached to opposite sides of nuclear envelope heterochromatin is highly condensed and not transcribed -includes DNA sequences that are generally not transcribed (highly repetitive sequences present at centromeres and telomeres)

Chromosomal domains

genomes are divided into discrete chromosomal domains: topologically associating domains (TADs) activity of an enhancer is specific for the promoter of its appropriate target gene -specificity is maintained by the organization of chromosomes within the nucleus into looped domains, formed by interaction of two molecules of CTCF and cohesion -enhancers can interact with promoters within the same looped domain, but are prevented from interacting with promoters in other domains Insulators --> divide chromosomes into independent domains & prevent enhancers from acting on promoters located in an adjacent domain -CTCF binds insulators

transcriptional activity of chromosomal domains correlates with their location in the nucleus (interphase)

heterochromatin is associated with the nuclear envelope or periphery of the nucleolus transcriptionally active chromatin is localized to interior or adjacent to nuclear pore complexes Gene-rich (active) chromosomes are found at center of nucleus -gene-poor (inactive) chromosomes found at nuclear periphery

protein splicing factor component play critical roles in spliceosome assembly

identification of the correct splice sites in pre-mRNA intron contains many sequences that resemble splice sites, so splicing machinery must be able to identify the appropriate 5' and 3' splice sites to produce functional mRNA splicing factors direct spliceosome to correct splicing site by binding to specific RNA sequences and then recruiting U1 and U2 snRNPs to the appropriate site on pre-mRNA by protein-protein interactions ex: SR splicing factors bind to specific sequences within exons and recruit U1 to the 5' splice site -also interact with U2AF, which binds to pyrimidine-rich sequences at 3' splice sites and recruits U2 to the branch point

proteins with NLS as recognized by nuclear transport receptors called importins

importins carry proteins through nuclear pore complex into the nucleus importins work with a GTP-binding protein called Ran, which controls the directionality of movement through the nuclear pore GTPase-activating protein (Ran GAP) associated with cytoplasmic filaments stimulate hydrolysis of GTP to GDP -in nucleus, Ran GEF (bound to chromatin) stimulates the exchange of GDP bound to Ran for GTP, leading to conversion of Ran/GDP to Ran/GTP and maintaining a high concentration of Ran/GTP in nucleus high concentration of Ran/GTP in nucleus determines directionality of nuclear transport

processing of mRNA in eukaryotes

in eukaryotes, mRNA synthesized in nucleus must first be transported to cytoplasm -pre-mRNA are extensively modified before export from nucleus -mRNA molecules are associated with proteins to form messenger ribonucleoprotein particle (mRNPs), which are responsible for mRNA processing, transport, and regulation of mRNA function processing of mRNA includes modification of both ends of initial transcript, and removal of introns from middle -processing reactions are coupled to transcription so mRNA synthesis and processing are closely coordinated steps in gene expression -C-terminal domain (CTD) of RNA polymerase II plays a key role in coordinating these processes by serving as a binding site for the enzyme complexes involved in mRNA processing (accounts for specificity in processing mRNAs) first step in mRNA processing is modification of 5' end of transcript by addition of 7-methylguanosine cap -added after transcription of first 20-30 nucleotides of RNA -initiates by addition of a guanine triphosphate (GTP) in reverse orientation to the 5' terminal nucleotide of the RNA -methyl groups added to this G residue and to the ribose moieties of one or two 5' nucleotides of the RNA chain -5' cap stabilizes the RNA and aligns eukaryotic mRNAs on the ribosome during translation most striking modification of pre-mRNAs is splicing -needs to be highly specific to yield functional mRNAs

alternative splicing

joining of exons in varied combinations to produce different mRNAs from the same gene -provides important mechanism for tissue-specific and developmental regulation of gene expression example is in sex determination in Drosophila -alternative splicing of same pre-mRNA determines whether a fly is male or female -alternative splicing of pre-mRNA of the gene transformer (tra) is controlled by protein SXL that is only expressed in female flies -Tra pre-mRNA has 3 exons, but a different second exon is incorporated into mRNA as a result of alternate 3' splice sites in two sexes -in males, exon 1 is joined to the most upstream of the 3' splice sites, which is selected by the binding of the U2AF splicing factor -in females, the SXL protein binds to the 3' splice site, blocking the binding of U2AF --> upstream 3' splice site is skipped in females, and exon 1 is joined to an alternate 3' splice site that is further downstream -exon 2 sequences in male tra mRNA contain a stop codon, so no protein is produces -stop codon is not included in female mRNA, so female flies express a functional tra protein, which acts as a key regulator of sex determination

in vitro splicing

key to understanding pre-mRNA splicing gene containing an intron is cloned downstream of a promoter recognized by bacterial virus RNA polymerase plasmid is digested with a restriction enzyme that cleave at 3' end to yield linear DNA molecule DNA is transcribed in vitro with the bacterial virus polymerase to produce pre-mRNA replacing reactions can be studied in vitro by addition of this pre-mRNA to nuclear extracts of mammalian cells

lamin assembly

lamins associate with each other to form higher-order structures -first, two lamins interact to form a dimer in which the α-helical regions of two polypeptide chains are wound around each other in a structure called a coiled coil -lamins associate with each other to form the filaments that make up nuclear lamina

amino acid is aligned on mRNA by complementary base pairing between mRNA codon and anticodon on tRNA

less stringent because of the redundancy of genetic code many amino acids attach to more than one species of tRNA 40 tRNAs that can recognize more than one codon in mRNA because of nonstandard base pairing (third base wobble) between the tRNA anticodon and the third position of a complemetnary codon G-U base pairs guanosine can be modified to inosine (can base pair with U, C, or A)

authophagy

lysosome are responsible for autophagy (turnover of cell's own components, including cytoplasmic organelles and cytosolic proteins) -ubiquitin-proteasome pathways is major mechanism of regulated protein degradation -authophagy leads to gradual degradation of long-lived components of the cell first step is enclosure of a small area of cytoplasm or a cytoplasmic organelle in a membrane derived from the ER -resulting vesicle (autophagosome) fuses with a lysosome, and its contents are digested uptake by most proteins in autophagosomes appear to be nonselective, so it results in the slow degradation of cytoplasmic proteins -some organelles can be selectively targeted for autophagic degradation (results of being marked by ubiquitylation) leads to continuous turnover of cellular constituents -can be regulated during development and in response to stress -plays important role in developmental processes -activated when cells are deprived of nutrients --> allows cells to degrade nonessential macromolecules so that their components can be reutilized -play important role in programmed cell death

at IRES, translation can initiate independently of the 5' cap

mediated by binding of IRES sequences directly to a reduced set of initiation factors (eIF4E is not involved) recognized directly by 40S ribosomal subunit recognized by eIF4G in complex with eIF4A, followed by recruitment of 40S subunit

histones modified by other modifications that occur at specific amino acid residues in histone tails and are associates with changes in transcriptional activity

methylation and phosphorylation of specific amino acid residues in histone tails, as well as by their acetylation histone modifications can affect gene expression by altering the properties of chromatin and by providing binding sites for other proteins that either activate or repress transcription alter condensation properties of chromatin methylation of H3K9 and H3K27 is associated with repression of chromatin condensation -methylated H3K9 and H3K27 residues serve as binding sites for proteins that induce chromatin condensation, linking histone modification to transcriptional repression and the formation of heterochromatin

Hereditary Nonpolyposis Colon Cancer (HNPCC)

mismatch repair system is important because mutations in MSH and MLH are responsible for HNPCC -researchers clones MSH and found that mutations in this gene were responsible for half of all HNPCC cases

excision repair

more general means of repairing a wide variety of chemical alterations to DNA damaged DNA is recognized and removed, either as free bases or as nucleotides -resulting gap is filled by synthesis of a new DNA strand, using the undamaged complementary strand as a template three types: -base-excision repair -nucleotide-excision repair: repair of most damage -mismatch repair: replication errors

transcriptional regulation in E.coli

most transcriptional regulation in bacteria operates at the level of initiation Example is lac repressor -β-galactosidase catalyzes the hydrolysis of lactose to galactose and glucose -only expressed when lactose is available -involved products of lactose permease (transports lactose into cell) and transacetylase (inactive toxic tiogalactosides) glucose is preferred energy source

recombinational repair

non-homologous end joining -can be repaired simply by rejoining the broken ends of a single DNA molecule --> high frequency of errors from deletion of bases around site of damage homologous recombination, which provides a mechanism for repairing damage and restoring the normal DNA sequence -in eukaryotes, only operative following DNA replication (newly replicated sister chromatids remain associated with each other) -breakage and rejoining of two parental DNA molecules, leading to reassortment of the genetic info of the two parental chromosomes -initiated at double-stranded breaks during DNA repair and recombination -both strands at break are resected by nucleases that digest DNA in 5' to 3' direction, yielding overhanging 3' single-stranded ends -single strands are bound by RecA in E.coli, and Rad51 in eukaryotes -RecA-DNA filaments bind a second double-stranded DNA molecule and catalyze the exchange of strands between homologous sequences

Nuclear bodies

nuclear bodies are a variety of discrete organelles -compartmentalize the nucleus and concentrate proteins and RNAs that function in specific nuclear processes -not enclosed by membranes but are maintained by protein-protein and protein-RNA interactions -can exchange contents with rest of the nucleus nucleolus --> function in rRNA synthesis, processing, and ribosome production Nuclear speckles --> storage of splicing components Polycomb bodies --> sites of polycomb proteins Cajal bodies --> sites of RNP assembly

processing of pre-RNA requires the action of proteins and RNAs that are localized to the nucleolus

nucleoli contains snoRNAs that acts as guide RNAs to enzymes responsible for pre-rRNA cleavage, ribose methylation, base modifications and pseudouridylation

protein import begins when an importin binds to the NLS of a cargo protein in cytoplasm

once cargo/importin complex reaches the nuclear side of envelope, Ran/GTP binds to importin, causing change in conformation of the importin, which disrupts the cargo/importin complex, displacing the cargo protein and releasing it into the nucleus cycle is maintained by export of the importin-Ran/GTP complex back through nuclear pore complex -in cytoplasm, GTP is hydrolyzed to GDP, which releases the importin -Ran/GDP is transported back to nucleus by its own import receptor (NTF2), where Ran/GTP is regenerated by action of Ran guanine-nucleotide exchange factor (Ran GEF)

important mechanism of epigenetic inheritance

patterns of DNA methylation are stably maintained following DNA replication by an enzyme that specifically methylates CpG sequences of a daughter strand in parental DNA, both strands are methylated at complementary CpG sequences --> replication --> parental strand of each daughter molecule is methylated --> newly synthesized daughter strands are methylated by an enzyme that recognizes CpG sequences opposite a methylation site gene that has been methylated and repressed in a parental cell remain methylated and repressed in progeny cell Methylation leads to stably inherited changes in gene expression: epigenetic inheritance

termination

polymerase remains associated with its template while it continues synthesis of mRNAs -unwinds template DNA ahead and rewinds DNA behind -with unwound portion, RNA chain is bound to complementary template DNA strand RNA synthesis continues until the polymerase encounters a termination signal -transcription stops, RNA is released from polymerase and dissociates from DNA template , and the enzyme dissociates from its DNA template -most common termination sequence in E.coli consists of a symmetrical inverted repeat of a GC-rich sequence followed by 7 A residues -transcription of GC-rich inverted repeat results in formation of a segment of RNA that forms a stem-loop structure by complementary base pairing ~disrupts RNA's association with the DNA template and terminates transcription

control of lac operon by glucose

positive control of transcription -effect of glucose on the expression of genes that encodes enzymes involved in breakdown of other sugars glucose repression is mediated by a positive control system -adenylyl cyclase (converts ATP to cAMP) is inhibited by α-ketoglutarate (intermediate in breakdown of glucose via the citric acid cycle) -when glucose is available, α-ketoglutarate is produced and adenylyl cyclase is inhibited -in absence of glucose, levels of α-ketoglutarate decrease, leading to activation of adenylyl cyclase and synthesis of cAMP -cAMP binds to transcriptional regulatory protein (CAP), which stimulates the binding of CAP to its target DNA sequences -CAP interacts with the α subunit of RNA polymerase, facilitating the binding of polymerase to the promoter and activating transcription

enhancers function by promoting binding transcription factors that regulate RNA polymerase

possible by DNA looping, which allows transcription factor bound to a distant enhancer (50 kb) to interact with proteins associated with Mediator, cohesion, and general transcription factor at the promoter loops are stabilized by cohesion transcription factors bound to distant enhancers can work by same mechanism as those bound adjacent to promoters

splicing of pre-mRNA

pre-mRNA is cleaved at the 5' splice site, and the 5' end of the intron is joined to an adenine nucleotide within the intron (near 3' end) -intron forms a loop next is simultaneous cleavage at the 3' splice site and ligation of the two exons intron is in a loop which is linearized and degraded within the nucleus of intact cells three critical sequence elements of pre-mRNAs -sequences at 5' splice site, 3' splice site, and sequences within the intron at the branch point

RNA polymerase

principle enzyme responsible for RNA synthesis catalyzes the polymerization of ribonucleotide triphosphates (NTPs) as directed by a DNA template does not require a performed primer to initiate synthesis of RNA -initiates de novo Five subunits -core polymerase -required for elongation -σ subunit is not required for the basic catalytic activity of the enzyme -σ subunit is required to identify the correct sites for transcription initiation -σ subunit required for promoter recognition

Formation of a polymerase II preinitiation complex in vitro

promoters of gene transcribed by polymerase II have different sequence elements -TATA box resembles the -10 sequence of E.coli promoters -initiator elements (Inr) ~TFIIB recognition elements (BRE) located upstream of start site ~DCE, MTE, anf DPE located downstream start site first step in formation of a transcription complex is binding of a general transcription factor TFIID to the promoter -composed of TATA-binding protein (TBP) and TBP-associated factors (TAFs) -TBP binds specifically to the TATA box, while other subunits of TFIID binds to the Inr, DCE, MTW, and DPE sequences -followed by recruitment of TFIIB, which binds to TBP and to BRE sequences ~serves as bridge to RNA polymerase II, which bonds to the TBP-TFIIB complex in association with TFIIF -binding of TFIIE and TFIIH completes formation of the preinitiation of complex ~two subunits of TFIIH and helicases, which unwind DNA around initiation site ~another subunit of TFIIH is a protein kinase that phosphorylates repeated sequences present in the C-terminus domain (CTD) ~consists of tandem repeats of seven amino acids with sequence TyrSerProThrSerProSer -phosphorylation of serine-5 in these CTD repeats by the TFIIH protein kinase releases the polymerase from its association with the preinitiation complex and leads to the initiation of transcription

chromatin remodeling factors

protein complexes that use energy-derived hydrolysis of ATP to alter contact between DNA and histones -alter arrangement or structure of nucleosomes catalyze sliding of histone octamers along the DNA molecule, repositioning nucleosomes to change the accessibility of specific DNA sequences to transcription factors may act by inducing changes in conformation of nucleosomes, affecting the ability of specific DNA sequences to interact with transcriptional regulatory proteins can eject histones from DNA, leaving nucleosome-free regions commonly found at enhancers and promoters can be recruited to DNA in association with transcriptional activators and can alter arrangement of nucleosomes to stimulate transcription

nuclear localization signal

proteins are targeted to the nucleus by specific amino acids sequences called nuclear localization signals (NLS), which are recognized by nuclear transport reactions -NLS first studied in simian virus 40 (SV40) T antigen: virus encoded protein that initiates viral DNA replication in infected cells -mutation of a single lysine residue prevents nuclear import, resulting in accumulation of T antigen in cytoplasm NLS are short stretched rich in basic amino acids residues (lysine and arginine) -amino acids that form the NLS are close together but not immediately adjacent -NLS of nucleoplasmin consists of Lys-Arg followed by 4 lysines located 10 amino acids downstream ~10 amino acids between can be mutated without affecting nuclear localization Specific peptide sequences serve as signals that direct proteins to the nucleus (or to other subcellular organelles)

glycosylation

proteins modified by addition of carbohydrates -proteins to which carbohydrate chain have been added (glycoproteins) are usually secreted or localized to the cell surface -carbohydrate moieties of glycoproteins play important roles in protein folding in the ER, in the targeting of proteins for delivery to the appropriate intracellular compartments, and as recognition sites in cell-cell interactions glycosylation of these cytoplasmic and nuclear proteins is thought to play a role in regulating their activities

chaperones

proteins that facilitate the folding of other proteins act as catalysts that facilitate assembly without being part of the assembled complex catalyze protein folding by assisting the self-assembly process function by binding to and stabilizing unfolded or partially folded polypeptides that are intermediates along the pathway leading to the final correctly folded state provide a isolated environment with which folding takes place ex: those that bind to nascent polypeptide chains that are still being translated on ribosome -chaperone binding stabilize amino (N) terminal portion in an unfolded conformation until the rest of the polypeptide chain is synthesized and then the completed protein can fold correctly

chaperones were initially identified as heat-shock proteins (Hsp)

proteins that have been subjected to elevated temperatures stabilize and facilitate the refolding of proteins that have been partially denatured as a result of exposure to elevated temperature Hsp70 chaperones and chaperonins act in a general pathway of protein folding in bacteria and eukaryotic cells -members of Hsp70 and chaperonin are found in cytosol and subcellular organelles of eukaryotic cells and bacteria Chaperones of Hsp70 bind to and stabilize unfolded polypeptide chains during translation and during transport of polypeptides into subcellular compartments -unfolded polypeptide is transferred from Hsp70 chaperones to a chaperonin, within which protein folding takes place, yielding a protein correctly folded into its functional 3-D conformation -ATP hydrolysis is required for release of the unfolded polypeptide from Hsp70 as well as for folding within the chaperonin chaperonin consists of multiple protein subunits arranged in two stacked rings to form a double-chambered structure -polypeptide chains are shielded from cytosol within chamber, and protein folding can proceed while aggregation is prevented

Electrophoretic-mobility shift assay

radiolabeled DNA fragment is incubated with a protein preparation and then subjected to electrophoresis through a nondenaturing gel protein binding is detected as a decrease in the electrophoresis mobility of the DNA fragment (migration is slowed by bound protein) use of electrophoretic-mobility shift assays a detailed mapping of protein-DNA interactions led to characterization of transcription factor binding sites binding sites are degenerate: transcription factor will bind not only to the consensus sequence, but also to sequences that differ from the consensus at one or more positions -physiologically significant regulatory sequences cannot be identified from DNA sequence alone

nucleotide-excision repair

recognizes a wide variety of damaged bases that distort the DNA molecule damaged bases are removed as part of an oligonucleotide containing the lesion in E.coli, UvrA recognizes damaged DNA and recruits UvrB and UvrC to the site of the lesion (UvrABC complex = excinuclease) -UvrB and UvrC cleave on the 3' and 5' sides of the damaged site, excising an oligonucleotide (12-13 bases) -helicase removes the damage-containing oligonucleotide, and resulting gap is filled by DNA polymerase I and sealed by ligase in mammalian cells, XPC recognizes the disrupted base pairing -XPA binds to the single-stranded DNA binding replication protein A (RPA) and a multi-subunit transcription factor (TFIIH), which is required to initiate transcription and contains the XPB and XPD helicases -XPB and XPD unwind the DNA, and the XPG and XPF/ERCC1 endonucleases are recruited and the DNA is cleaved, excising the damaged oligonucleotide -gap is filled by DNA polymerase δ and sealed by ligase

base excision repair

repairs uracil-containing DNA excision of uracil is catalyzed by DNA glycosylase (enzymes that cleaves the bond linking uracil to the deoxyribose of the DNA backbone - recognizes specific forms of damaged bases) yields a free uracil and an apyrimidinic site (sugar with no base - AP site) sites are repaired by AP endonuclease, which cleaves adjacent to the AP site remaining deoxyribose moiety is removed by deoxyribosephosphodiesterase, and the resulting single-base gap is filled by DNA polymerase and ligase

transcription in eukaryotic cells is regulated by repressors as well as activators

repressors bind to specific DNA sequences and inhibit transcription could interfere with binding of other transcription factors to DNA ex: binding of repressor near transccription start site -can block interaction of RNA polymerase or general transcription factors with promoter repressors contain same DNA binding domain as activators but lack activation domain actively repress: -inhibit transcription through direct interaction -recruit corepressors many repressors (active repressors) contain specific functional domains that inhibit transcription via protein-protein interactions -regulators in cell growth and differentiation -inhibit transcription by interacting with specific activator proteins, with Mediator proteins, or general transcription factors and with corepressors

initiation of translation in eukaryotes

require 12 proteins designated eIFs -eIF2 (in complex with GTP) binds to the initiator methionyl tRNA, which forms a complex with 40S -5' cap of mRNA is recognized by eIF4E, and the eIF4 initiation factors bring the mRNA to the 40S ribosomal subunit, which scans the mRNA to identify the AUG initiator codon (scanning requires energy from ATP hydrolysis) -eIF5 triggers hydrolysis of GTP - eIF2 -initiation factors are released, and eIF5B facilitates binding of 60S subunit to form 80S initiation complex of eukaryotic cells

rRNA catalyzes peptide bond formation

ribosomal proteins were absent from the site at which the peptidyl transferase reaction occurred ribosomal proteins play a structural role

elongation stage of translation

ribosome has 3 sites for tRNA binding: P(peptidyl), A (aminoacyl), and E (exit) sites -initiator methionyl tRNA is bound at P site -aminoacyl tRNA is escorted to ribosome by an elongation factor (eEF1α), which is complexed to GTP, to the A site, pairing with the second codon of the mRNA -when eEF1α leaves the ribosome, a peptide bond forms between methionyl tRNA at the P site and the aminoacyl tRNA at the A site (catalyzed by large ribosomal subunit) -methionine is transferred to the aminoacyl tRNA at the A site, forming a peptidyl tRNA and leaving the uncharged initiator tRNA at the P site -translocation requires eEF2 and is coupled to GTP hydrolysis: ribosome moves 3 nucleotides, positioning next codon in empty A site -binding of a new aminoacyl tRNA to A site induces release of uncharged tRNA from E site accuracy is not accounted for by base pairing alone -decoding center in small ribosomal subunit, which recognizes correct codon-anticodon base pairs and discriminates against mismatches -correct base pairing triggers a conformational change that induces the hydrolysis of GTP bound to eEF1α and release of the elongation factor bound to GDP

nucleolus is site of ribosome assembly

ribosome production factory, designed to fulfill the need for regulated and efficient production of rRNAs, and assembly of the ribosomal subunits formation of ribosome involves the assembly of the ribosomal precursor RNAs with ribosomal proteins and 5S rRNA -ribosomal proteins are transported from cytoplasm to the nucleolus, where they are assembled with rRNAs to form pre-ribosomal particles -5S rRNAs are assembled into pre-ribosomal particles within the nucleolus -pre-ribosomal particles exported to cytoplasm, forming active 40S and 60S subunits of eukaryotic ribosomes

mismatch repair

scans newly replicated DNA enzymes identify and excise the mismatched base specifically from the newly replicated DNA strand to less than 10^-9 in E.coli, newly synthesized strands are not methylated -it is initiated by protein MutS, which recognizes the mismatch and forms a complex with two other proteins called MutL and MutH -MutH endonuclease cleaves the unmethylated DNA strand opposite a methyl group -MutL and MutS act with an exonuclease and helicase to excise the DNA between strand break and mismatch -gap filled by DNA polymerase I and ligase Eukaryotic cells don't have homolog of MutH, and strand specificity isn't determined by DNA methylation -presence of single-strand breaks in newly replicated DNA appears to specify strand to be repaired -MSH (MutS) and MLH (MutH) bind to the mismatches base and direct excision of the DNA between the strank break and mismatch -lagging strand could be identified by nicks at either end of Okazaki fragments, and leading strand can be identified by its growing 3' end -DNA pol ε replicated leading strand, while DNA pol δ replicated lagging strand

regulation of translation by miRNAs

siRNAs generally pair perfectly with their targets and induce cleavage of mRNA by RISC, but most miRNAs form mismatched duplexes with sequences within the 3' untranslated regions of their target mRNAs miRNAs associate with the RISC complex two miRNA strands are unwound and RISC is targeted to the 3' untranslated region (UTR) of an mRNA, leading to inhibition of translation, deadenylation, and mRNA degradation

the ribosome

site of protein synthesis -two distinct subunits, each containing protein and rRNAs in prokaryotes, there is a large and small subunit -each ribosome contains one copy of rRNAs and one copy of each of the ribosomal proteins subunits of eukaryotic ribosomes are larger and contain more proteins ribosomes can be formed in vitro by self-assembly of their RNA and protein constituents -ribosomal proteins and rRNAs can be mixed and will re-form a functional ribosome

some proteins in eukaryotic cells are modified by attachment of lipids to the polypeptide chaine

target and anchor these proteins to the plasma membrane, with which the hydrophobic lipid is able to interact (either inner or outer face of plasma membrane) carbohydrates added in the ER and Golgi exposed on cell surface

the nuclear lamina

the association of lamins with the inner nuclear membrane is facilitated by the posttranslational addition of lipids -lamins bind to specific inner nuclear membrane proteins and are directly connected to the cytoskeleton by protein complexes (LINC) that span the inner and outer nuclear membranes -lamins can also bind to chromatin (important in gene expression) localization of heterochromatin to periphery

nuclear pore complexes

the channels through which small polar molecules, ions, and macromolecules (proteins and RNA) can travel between nucleus and cytoplasm -extremely large -composed of multiple copies of 30 different pore proteins (nucleoporins) plays fundamental role in physiology of eukaryotic cells -RNA synthesized in nucleus must be efficiently exported to cytoplasm -proteins for nuclear function (transcription factors) must be transported to nucleus from cytoplasm -many proteins shuttle continuously between nucleus and cytoplasm most proteins and RNAs pass through the nuclear pore complex by selective transport -recognized by specific signals that direct their transport -determine composition of nucleus and plays critical role in regulating transcription

proteins anchored to the outer face of the plasma membrane are modified in the ER by addition of lipids linked to oligosaccharides (glycolipids) to their C terminus

this structure is called a glycosylphosphatidylinositol (GPI) anchor GPI anchors are joined to the C-terminal amino acid by ethanolamine glycolipid is joined to the inositol head group of phosphatidylinositol

Eukaryotic RNA polymerases

three distinct nuclear RNA polymerases (I, II, and III) protein-coding genes are transcribed by RNA polymerases II to yield mRNAs -also transcribe microRNAs and long noncoding RNAs polymerases I and II transcribe rRNAs and tRNAs -RNA polymerase III also transcribes snRNAs and scRNAs, while others are polymerase II transcripts general transcription factors and initiation of transcription by RNA polymerase II -transcription in eukaryotic system appeared to require distinct initiation factors that were not associated with the polymerase -general transcription factors are involved in transcription from most polymerase II promoter ~control expression of individual genes, and thus responsible for regulating gene expression

The process of translation

three stages: initiation, elongation, and termination initiation starts with binding of a specific initiator methionyl tRNA and mRNA to small ribosomal subunit -large ribosomal subunit then joins single mRNA can be translated simultaneously by several ribosomes -group of ribosomes bound to mRNA is called a polyribosome/polysome

regulation of nuclear protein import

transcription factors are functional only when they are present in the nucleus, so regulation of their import to nucleus controls gene expression one mechanism of regulation: transcription factors associate with cytoplasmic proteins that mask their NLS -because signals aren't recognizable, proteins remain in cytoplasm Ex: NF-xB, which is activated in response to extracellular signals -in unstimulated cells, IxB is bound to NF-xB, inhibiting it from being transported into the nucleus -in stimulated cells, IxB is phosphorylated a degraded, allowing NF-xB to enter the nucleus and activate transcription nuclear import of other transcription factors is regulated directly by their phosphorylation -ex: Pho4

transcription factories

transcription occurs at clustered sites (transcription factories) that contain newly synthesized RNA and are highly enriched in active RNA polymerases and transcription factors -transcription factories contain 8 molecules of RNA polymerase -active genes loop out and are pulled through a factory as transcription occurs -transcription factories increase efficiency of gene expression and facilitate the coordinated regulation of related genes

direct link between histone acetylation and transcriptional regulation

transcriptional activators and repressors are associated with histone acetyltransferase (HAT) and deacetylases (HDAC) - coactivators and repressors -revealed by cloning a gene encoding a HAT from tetrahymena -sequence of HAT was closely related to Gcn5p (yeast transcriptional coactivator) -Gcn5P has HAT activity, suggesting that transcriptional activation results from histone acetylation -HATs are associated with mammalian transcriptional coactivators and general transcription factor TFIID -corepressors function as histone deacetylases (remove acetyl groups from histone tails) histone acetylation plays a key tole in regulation of eukaryotic gene expression

tRNAs

translation is carried out on ribosomes, with tRNAs serving as adaptors between the mRNA template and amino acids being incorporated into protein all cells have a variety of tRNAs that serve as adaptors -possess unique identifying sequences that allow correct amino acids to be attached all have the sequence CCA at their 3' terminus -amino acids are covalently attached here -mRNA template is recognized by the anticodon loop, which binds to the appropriate codon on mRNA by complementary base pairing

regulation of translation

translation of mRNAs can be regulated by translational repressor proteins and noncoding microRNAs

The difference in protein synthesis of prokaryotic and eukaryotic cells are in the signals that determine the position at which synthesis is initiated

translation starts at specific initiation sites 5' terminal portion of mRNA are noncoding sequences known as 5' untranslated regions (UTR) eukaryotic mRNAs usually encode a single polypeptide chain (monocistronic) prokaryotic mRNAs encode multiple polypeptides (polycistronic) both eukaryotic and prokaryotic mRNAs end in noncoding 3' UTRs

enzyme regulation be protein-protein interactions is cAMP-dependent protein kinase

two regulatory and two catalytic subunits -in this state, the enzyme in inactive because the regulatory subunits inhibit enzymatic activity of the catalytic subunits -enzyme is activates by cAMP, which binds to the regulatory subunits and induces a conformation change, leading to dissociation of the complex -free catalytic subunits are enzymatically active protein kinases cAMP acts as an allosteric regulator by altering protein-protein interactions

immunoglobulins

type of antibody -very diverse, enabling different antibodies to recognize a vast array of foreign antigens -encoded by unique lymphocyte gene that are formed during development of the immune system as a result of programed rearrangements consist of heavy and light polypeptide chains -both composed of C-terminal constant regions and N-terminal variable regions each B lymphocyte only produces a single type of antibody -each antibody is encoded by unique genes formed by site-specific recombination during B lymphocyte development