Cell Biology Exam 2

modification of histones

- DNA is wrapped tightly around histones which makes it difficult for RNA polymerase and transcription factors to bind - basic unit of chromatin is the nucleosome. core histones have two domains. - amino terminal tail domain is subject to several types of covalent modifications that influence transcription: 1. acetylation of lysine residues = lysine amino acids are pos. charged, therefore interacting with neg. charged DNA (leads to tight packing). when acetylated, the charge is neutralized and histones fall off, causing DNA to relax and become more available for RNAP to bind 2. histones can also be modified by methylation of lysine and arginine, phosphorylation of serine, and addition of small peptides to lysine. occurs at specific amino acid residues to change the nature of the DNA.

DNA proofreading likely provided the evolutionary pressure for:

- DNA polymerase dependency on an annealed primer - 5' to 3' directionality of synthesis

ENCODE project (KEY EXP)

- ENCODE project was launched with the goal of defining all of the functional elements in the human genome. - involved analysis of human cell lines using a variety of different methods. - most striking finding was that almost all of our genome, at least 80%, has a function that could be characterized by this genomic analysis. - about 75% of the genome was transcribed into RNA - uncovered a new way of looking at mammalian genomes = not majority junk DNA, most of it is functional in some way, extent to which noncoding RNAs regulate gene expression may be much greater than previously appreciated

Enzymes involved in eukaryotic DNA replication:

- Primase forms RNA primer for both leading and lagging strand leading strand synthesis: - as primase makes primer, DNA polymerase alpha begins polymerization and then DNA polymerase epsilon finishes out majority of polymerization - RNase H removes the primer by 5' to 3' exonuclease activity and degrades the RNA primer - DNA polymerase delta fills in the gap - DNA ligase joins the fragments lagging strand synthesis: - as primase makes primer, DNA polymerase alpha begins the okazaki fragment (slow enzyme) - DNA polymerase delta takes over finishing the base pairing (faster enzyme), it does most of the work on lagging strand. - RNase H removes the primer by 5' to 3' exonuclease activity and degrades the RNA primer - DNA polymerase delta fills in the gap - DNA ligase joins the fragments

Enzymes involved in prokaryotic DNA replication:

- Primase forms RNA primer for both leading and lagging strand - DNA polymerase III carries out polymerization on both the leading strand and the lagging strand. it is one of most efficient DNA polymerases, can replicate DNA very quickly (bacteria copied ~20 minutes) - DNA polymerase I has both 5' to 3' exonuclease activity and 5' to 3' polymerase activity. it removes the primer and fills in the gap. - DNA ligase joins the fragments

transcriptional repressors

- act via protein-protein interacts with general transcription machinery (i.e. mediator protein), which blocks activation/elongation of transcription by RNAPII - some act by blocking binding of transcriptional activators or general transcriptional machinery (compete for same binding site); sometimes referred to as active repressors

chromatin structure and transcription:

- actively transcribed genes are located in euchromatin (mostly 30nm fibers) - regulation of physical accessibility of a gene to the machinery and/or process of transcription is another mechanism of transcription regulation two modes: 1. modification of histones 2. rearrangement of nucleosomes via chromatin remodeling factors - histone modifying enzymes (HATs, HDACs) and chromatin remodeling factors bind to the phosphorylated CTD of RNAPII, modulating the chromatin structure as RNAPII transcribes

fundamental principles of cells

- each cell has a life of its own - there are about 50 trillion cells in the human body (each one has 3 billion base pairs) - cells are dynamic, living units that respond to their environments - cells have specialized functions - cells must cooperate and communicate with each-other for the sake of the organism - all cells arise from pre-existing cells (true for ALL cells)

RNA polymerase in eukaryotes:

- eukaryotic cells have three RNA polymerases that transcribe different classes of genes; they must interact with additional proteins to initiate and regulate transcription; chromatin structure regulation is important in gene expression 1. RNAP II = transcribes ALL protein-coding genes, miRNAs, lncRNAs 2. RNAP I = transcribes rRNA (28S, 18S, 5.8S) 3. RNAP III = transcribes tRNAs, rRNA(5S), snRNAs there is also an RNAP that is unique to mitochondrial DNA

the discovery of introns (KEY EXP)

- first discovered during studies of the replication of adenovirus. - processing steps of primary mRNA transcripts thought to involve the removal of sequences from 5' and 3' ends. - used adenovirus 2 to investigate mRNA synthesis in human cells. (advantage of adenovirus 2 = viral DNA can be isolated directly from virus particles and mRNAs encoding viral structural proteins are present in such high amounts that they can be purified directly from infected cells) - focused on an abundant mRNA that encodes a viral structural protein known as HEXON. - in order to map the hexon mRNA on viral genome, purified mRNA was hybridized to adenovirus DNA and hybrid molecules were examined. Under microscope, found that the body of the hexon mRNA formed hybrids with restriction fragments of adenovirus that had previously been known to contain the hexon gene. sequences at 5' end failed to hybridize to DNA sequences adjacent to those encoding the body of the gene (suggested that 5' end of mRNA had arisen from sequences located elsewhere in genome) - tested possibility by hybridization of hexon mRNA to restriction fragment extending upstream of hexon gene. mRNA-DNA hybrids formed displayed a complex loop structure. 5' end of hexon mRNA hybridized to three short upstream regions of DNA, separated from eachother and the body of the gene by large single stranded DNA loops (introns)

Ex. positive control in lac operon

- glucose is the preferential source of energy - E. coli will express beta-galactosidase only if glucose is NOT available (in order to breakdown lactose to make glucose) - presence of glucose represses expression of the lac operon even if lactose is also present - E. coli links the expression of the lac operon to glucose levels through catabolite activator protein (CAP) which recruits RNA polymerase to lac operon promoter - CAP must be bound to cAMP Low glucose -> adenylyl cylcase cyclizes ATP to make cAMP -> cAMP binds to CAP which stimulates binding to target DNA sequence upstream and facilitates binding of RNAP to promotor -> transcription occurs

Ex. cAMP-dependent protein kinase

- has 4 subunits: two regulatory and two catalytic subunits in the inactive form - cAMP binds to regulatory subunits which induces conformational change and dissocation of the complex. - free catalytic subunits are now enzymatically active protein kinases - cAMP acts as an allosteric regulator by altering protein-protein interactions

Ex. Lac operon in E. coli

- lac operon = a set of genes expressed as a unit that encodes enzymes that carry out lactose metabolism. in bacteria there are no introns so genes can be directly adjacent to eachother and be transcribed as a unit (= operon) - operator = sequence in which regulatory proteins can bind - lactose metabolism involves specific enzymes. the lac operon encodes three of them that are only expressed when lactose is present: 1. beta-galactosidase = cleaves lactose into glucose and galactose 2. lactose permease = transports lactose into the cell 3. transacetylase = inactivates toxic thiogalactosides that are transported into the cell along with lactose not efficient use of energy to make protein/enzymes if lactose doesn't need to be broken down. lac operon is under both negative and positive control

cellular roles of introns:

- many introns encode functional products, which may be either proteins or non coding RNAs. nested genes = one gene is contained within an intron of a larger gene. transcription yields primary transcripts of both the host and nested genes, which are spliced to yield host gene and nested gene mRNAs. - some introns contain regulatory sequences that control gene expression. regulatory elements that control transcription may be located up to hundreds of kilobases away from the gene, immediately upstream, within noncoding exons or introns (UTRs). - introns allow for alternative splicing = differential inclusion/exclusion of exons during splicing, yielding multiple possible mature mRNAs from the same gene (make different proteins from the same gene). on average, each human gene yields 5 alternative transcripts.

covalent modifications of proteins:

- phosphorylation - acetylation of lysine - methylation of lysine and argenine - nitrosylation (addition of NO groups) to cysteine - glycosylation of serine and threonine

prokaryotic vs eukaryotic mRNAs

- prokaryotic mRNA is poly-cistronic = encodes multiple proteins, each of which is translated from an independent start site. there are multiple start sites. - eukaryotic mRNA is mono-cistronic = encodes a single protein, only one start site in both cell types, translation always starts with methionine (usually AUG). but the signals that identify initiation are different. prokaryotes: shine-dalgarno sequence = AGGAGG = precedes AUG start codon in bacterial mRNAs; aligns mRNA on the ribosome because recognized by small subunit eukaryotes: small subunit recognizes 5' cap and then ribosomes can until reach initiation codon

what roles do snRNP snRNAs play?

- recognize and align the snRNPs at the branch point and splice sites - directly catalyze the splicing reaction (can still catalyze reaction of cutting if get rid of all protein in spliceosome) formation of loop and excision of intron can be catalyzed by U2 and U6 snRNAs in absence of protein (catalyze removal of their own introns)

Ex. negative control in lac operon

- repressor protein = slows down or stops transcription by binding to the operator region (right in front of promoter); blocks RNAP from binding - repressor protein binds to operator sequence only when lactose is not present (no substrate to break down = no need to make enzyme) - when lactose is present in normal cells, it binds to the repressor, preventing it from binding to the operator and the genes are therefore expressed. - lactose regulates the repressor protein and the repressor protein regulates transcription

translational regulation of specific mRNAs

- some translational repressors bind to specific sequences in the 3' UTR. - some bind to initiation factor eIR4E, interfering with its interaction with other initiator factor proteins and inhibits the initiation of translation (if all initiation factors cannot all be assembled together, translation cannot start) - modulation of the length of the poly-A tail affects translation rates = proteins bind 3'UTR and either shorten tail temporarily to inhibit translation or lengthen tail when translation is needed. - RNA interference = mediated by short dsRNAs, it is used as an experimental tool to block gene expression at the level of translation. mediated by siRNAs and miRNAs.

human telomeres and telomerase

- telomerase activity maintains telomeres at their normal length - most somatic cells don't have enough telomerase to maintain telomere length for an indefinite number of cell divisions - telomeres gradually shorten as cells age, which eventually leads to cell death or senescence = can't divide anymore - several premature aging syndromes are characterized by abnormally high rates of telomere loss; some are caused by mutations in telomerase - cancer cells express abnormally high levels of telomerase allowing them to continue dividing indefinitely

wobble hypothesis

- the base in the first position on tRNA (5' end) is usually an abnormal base like inosine, pseudoeuridine, tyrosine, etc. - these abnormal bases can pair with more than one type of nitrogenous base in the third position of the codon of the mRNA - problem = determine anticodon (tRNA sequence) for any given mRNA

translational regulation

- translational regulation of specific mRNAs via translational repressor proteins, regulated polyadenylation, RNA interference with miRNAs - global regulation of translation through regulation of initiation factors eIF2 and eIF4E - regulation of where specific mRNAs are translated via mRNA localization

transcriptional activators

- two domain structure (proteins can have domains that have own tertiary structure and specific function.activity within the entire protein) -> one domain binds the DNA specific to that TF, second domain activates transcription - some transcriptional activators act via protein-protein interactions with general transcription machinery (i.e. mediator protein) = second domain stimulates transcription through these interactions by pushing polymerase to work faster either by facilitating better RNAP recruitment or better activation of RNAP

translational regulation and early development

- very important during early development - many mRNAs with short poly-A tails are stored in oocytes; translation activated at fertilization or later stages. - lengthening poly-A tails allows binding of poly-A binding protein which stimulates translation

mRNA processing steps

1. 5' capping 2. 3' polyadenylation 3. splicing

3 stages of translation

1. Initiation 2. Elongation 3. Termination

4 types of lipid modifications to proteins:

1. N-myristolation(end up on inside plasma membrane) 2. prenylation(end up on inside plasma membrane) 3. palmitoylation (end up on inside plasma membrane) 4. glycolipids (end up on outside plasma membrane)

transcription in prokaryotes:

1. RNAP bound nonspecifically to DNA, scanning it. 2. RNAP recognizes -35 and -10 promoter sequences and sigma subunit binds to unwound DNA (closed-promoter complex) 3. unwinding of DNA around initiation site = open-promoter complex (only transcribes 3' to 5' strand) 4. initiation of transcription 5. sigma subunit is released once ~10 nucleotides transcribed 6. elongation of RNA chain by RNAP 7. RNAP dissociates when reaches termination signal

what are the 5 general transcription factors?

1. TFIID 2. TFIIB (recruitment of RNAPII) 3. TFIIF (recruitment of RNAPII) 4. TFIIE 5. TFIIH (important)

how does attachment of amino acid to tRNA occur?

1. amino acid is joined to AMP, forming aminoacyl AMP 2. amino acid is transferred to the 3' CCA end of the tRNA and AMP is released *ATP dependent process* charged tRNA = aminoacyl tRNA = amino acid attached cells have about 40 different tRNAs for the 20 different amino acids.

What are the three main types of repair mechanisms?

1. mismatch repair 2. base-excision repair 3. nucleotide excision repair

what was the early evidence for the physical structure of chromatin?

1. nuclease digestion of genomic DNA yielded fragments of ~200 bp increments 2. electron microscopy of genomic DNA revealed a 'beads on string' structure nucleosome core responsible for ladder pattern on gel electrophoresis because DNA can only be cut within each particle which are in intervals of about 200 bp apart. conclusion = chromatin structure consists of 200bp intervals of DNA in complex with proteins, proteins protect DNA from being cut.

what are the cis-acting elements in transcriptional regulation of eukaryotic genes?

1. promoter sequence 2. upstream regulatory sequences 3. enhancer sequence

what are the types of repetitive sequences?

1. simple sequence repeats = tandem (one after the other) arrays of up to thousands of copies of short sequences. satellite DNAs = consists of very large arrays of tandemly repeating, non-coding DNA. do not encode proteins but play important roles in chromosome structure. 2. short interspaced elements (SINEs) and long interspaced elements (LINEs) = transposable elements (capable of moving to different sites in genomic DNA), they are retrotransposons; transcription is mediated by reverse transcription in which an RNA copy of a SINE or LINE is converted to DNA by reverse transcriptase within the cell. 3. retrovirus like elements = move within the genome by reverse transcription. 4. DNA transposons = interspersed repetitive elements that move through the genome by being copied and reinserted as DNA sequences, rather than move by reverse transcription.

what do chaperones do?

1. stabilize unfolded polypeptide chains to prevent aggregation -- critically important within cell because of high concentration of other proteins - chaperones bind to hydrophobic regions (hydrophobic portions are the areas that will want to clump/interact) - i.e. heart shock protein -70 (hsp70) = upregulated when stress cell with elevated temperature in order to stabilize and facilitate refolding of proteins that have been partially denatured - bind to polypeptide chains that are still being translated on the ribosome. the chain must be protected from aberrant folding or aggregation with other proteins until synthesis of an entire domain is complete. - stabilize unfolded polypeptide chains during transport into organelles 2. provide an isolated environment within which correct folding takes place. this is carried out by chaperonins = provide an isolated environment within which correct folding takes place. - ATP driven process, needed to get protein into and out of chaperonin - act in concert with hsp70 chaperones to facilitate folding of newly translated proteins - consist of subunits arranged in two stacked rings to form a double chambered structure. this isolates protein from cytosol and other unfolded proteins. - folding takes place within chaperonin

5' capping

5' end of pre-mRNA transcript is modified by addition of a 7-methylguanosine cap (catalyzed by guanylyl transferase) 5' cap stabilizes the RNA and aligns it on the ribosome during translation (mediates interactions with ribosomes) exonucleases are prevented from degrading the pre-mRNA

promoter sequence in bacteria:

6 nucleotides long, located at -10 and -35 bp upstream of TSS. sigma subunit identifies these sites to begin transcription.

mismatch repair

= a DNA repair mechanism by which cells can repair mismatched base pair incorporated during DNA replication; if DNA polymerase did not catch mistake with 3' to 5' exonuclease activity, there is mismatch repair mechanism to fix mistake. carried out by three proteins in E. coli: 1. MutS recognizes the mismatch in DNA and forms a complex with MutL and MutH 2. MutH cleaves the new strand adjacent to the mismatch; cleaves opposite a methyl group (at a GATC sequence); MutH = endonuclease 3. Mut S and MutL direct excision between nick and mismatch; entire piece is cut out and DNA polymerase and ligase fill it in again in eukaryotes: not dependent on methylation so MutH is not present. Only MutL and MutS. single strand breaks in newly replicated DNA (ends of growing strands) appears to specify the strand to be repaired. MutS and MutL bind to mismatche dbase and direct excision of the DNA between a strand break and the mismatch.

phosphorylation

= covalent attachment of a phosphate group, common event by which cells regulate proteins mechanisms: can alter a protein's conformation, can alter a protein's interaction with other molecules serine, threonine, tyrosine are the three amino acids that can be phophorylated (b/c of hydroxyl OH groups) catalyzed by kinases = proteins that transfer phosphate groups from ATP to hydroxyl groups of side chains of serine, threonine, and tyrosine phosphorylation is reversible; it can activate or inhibit proteins in response to environmental signals it is revered by protein phosphatases = catalyze hydrolysis of phosphorylated amino acids. i.e. regulation of eIF2 and eIF2B by phosphorylation in response to cell stress i.e. binding of transcription elongation and processing factors to phosphorylated CTD of RNAPII (TF2H catalyzes this phosphorylation)

Elongation

= polypeptide chain elongates by successively adding amino acids, requires several eukaryotic elongation factors (eEFs) ribosomes have 3 binding sites: P (peptidyl), A (aminoacyl), E (exit) initiator methionyl tRNA binds to the P site. the next aminoacyl tRNA binds to the A site by pairing with the second codon of the mRNA. eEF1 alpha = elongation factor coupled to GTP that brings the aminoacyl tRNA to the ribosome. Loading into A site of ribosome requires energy (GTP that is bound to the EF) once two tRNAs are in ribosome (one in P and one in A), peptide bond can be formed. mRNA is then translocated (moves along through ribosome) to be read. this requires GTP bound eEF2. once tRNA is in E site, it is lost and recycled.

nucleotide excision repair

= removes damaged bases as part of an oligonucleotide useful in UV light damaged DNA (dimers) and carcinogenic substance damage excinuclease = an enzyme complex that can directly excise an oligonucleotide (includes the mutation) helicase is required to unwind the DNA for excision; resulting gap is filled by DNA polymerase and sealed by ligase

initiation of translation

= ribosome binds at 5' UTR and initiates polypeptide synthesis at the start codon. large subunit only gets involved once AUG is recognized. this is when ribosome fully built together. begins with small subunit recognizing the 5' cap and then scanning mRNA for start codon. translation initiation requires several eukaryotic initiation factors (eIFs): - eIF2 = carries charge tRNA methionine bound to GTP - PABP = binds poly-A tail - eIF4E = binds 5' methyl cap scanning process is energy dependent. for each codon scanned, ATP is used. Once start codon is recognized, GTP hydrolyzed to GDP and large subunit binds.

base exision repair

= single damaged bases are recognized and removed (only used for broken nucleotides) multiple enzymes are involved: - DNA glycosylase = recognizes the defect and cleaves the bond between the base and the deoxyribose of the DNA, forming a AP site (sugar with no base attached) - AP endonuclease = repairs these sites. deoxyribose is removed and resulting gap is filled by DNA polymerase and ligase *good for uracil containing DNA repair*

ubiquitin-proteasome pathway

= targeted degradation of proteins by the proteasome proteasome = large, multi-subunit protease complex; has multiple enzymes on the inside which chew up/break down the protein into smaller peptides ATP dependent process to get protein into proteasome. ubiquitin = small protein that targets the proteins for degradation. it is attached to the amino group of the side chain of a lysine residue, and then more are added to form a chain. proteasome recognizes and degrades only ubiquinated proteins. more ubiquinated groups attached = higher likelihood for proteasome to recognize and accept.

termination

= when a stop codon is encountered, polypeptide is released and ribosome dissociates release factors (RFs) recognize these signals and terminate protein synthesis. fit right into the A site. in prokaryotic cells, RF1 recognizes UAA or UAG; RF2 recognizes UAA or UGA. in eukaryotic cells, eRF1 recognizes all three stop codons

replication fork

A Y-shaped region on a replicating DNA molecule where new strands are growing

processed pseudogene

An inactive gene copy which lacks introns and the normal sequences that direct transcription. mRNA, lacking introns, copied by reverse transcription, yielding a cDNA copy lacking introns. integration into chromosomal DNA results in processed pseudogene.

how do enhancers regulate a gene from an alternate location?

DNA is a 3-dimensional structure that loops around. DNA looping enables enhancers to function at long distances from TSS DNA looping allows transcription factor bound to distant enhancer to interact with proteins associated with the RNA polymerase/mediator complex at the promoter enhancers are specific to genes, DNA structure is not random = needs to be organized in specific way for all these sequences to work.

how does DNA replication end?

DNA polymerase cannot copy the 3' end of the lagging strand template.... when primer is removed, it ends up missing some DNA because DNA polymerase cannot fill it in (only polymerize in 5' to 3' and no primer available). this is an issue because our chromosomes would get shorter over multiple cell replications. to combat this issue, old/template strand is extended in order to provide space for primase to create an RNA primer for DNA polymerase (when primer ultimately cut off, won't matter because already replicated necessary portion) this is where telomeres and telomerase come in to play

how is DNA polymerase loaded and stabilized on the ssDNA template?

DNA polymerase does not hang out by itself, it needs to get locked and held in place so that it can work faster sliding clamp proteins = function to load the polymerase on to the primer and maintain stable association with the DNA template clamp-loading proteins = use energy from ATP hydrolysis to open the sliding clamps and load them on to the DNA template

DNA polymerase and mutation rates

DNA polymerase helps select the correct base for insertion. binding of the correct nucleotide induces conformational changes in DNA polymerase that leads to incorporation of the nucleotide. DNA polymerase makes mistake about every 100,000 bp (without proofreading ability) proofreading by DNA polymerase reduces error rate to mistake about every 1,000,000-100,000,000,000 bp.

semi-conservative replication

DNA replicates semiconservatively in which one strand is used as template for new DNA strand. New DNA molecule consists of old strand (template strand) and new strand

How does DNA replication begin?

DNA replication happens at a replication fork but does not start at a replication fork. DNA replication starts at origins of replication (ori) origins of replication = binding sites for proteins that initiate the replication process. it is a sequence of bp in which initiate proteins bind. initiator proteins need to get involved first and recognize ori before RNA primer is synthesized and DNA gets unwinded by helicase/SSBP process of replication begins in both direction of the ori (2 helicases, one on each side; multiple ss binding proteins); two replication forks are formed

cis-acting elements

DNA sequences in vicinity of a gene that regulate its expression (i.e. operator)

chromatosome

DNA wrapped around histone core particle and bound by linker histone H1

example: transposition of retrogene determines short legs in dog breeds

Fgf4 gene = fibroblast growth factor 4, both big dogs and small dogs have this gene but presentation in genome is different. - big dogs have normally distributed Fgf4 gene. in little dogs, the gene has been transposed into a different piece of DNA. it lacks introns and sits in the middle of two LINE sequences. the LINE sequence does not contain promotor sequences which allow for normal transcription and expression of the gene, which results in premature termination of bone growth and therefore, short legs.

what are the 5 major types of histones?

H1, H2A, H2B, H3, H4 extremely abundant proteins in eukaryotic cells, together their mass is about equal to that of the cell's DNA

untranslated regions

Located in mRNAs and appear at 3' and 5' ends, have roles in regulation of translation.

termination of transcription in prokaryotes:

RNA polymerase stops transcribing when it reaches a termination signal. termination signal= adjacent complimentary sequences located at the end of the genes transcription of GC-rich inverted repeat results in a segment of RNA that forms a stable stem-loop structure, which disrupts RNAP association with the DNA template and terminates the transcription. RNAP cannot get past the termination signal sequence because falls off when stem loop structure forms. transcription of some genes is terminated by Rho = specific terminator protein which binds extended segments of single stranded RNA

primase

RNA polymerase that synthesizes short fragments of RNA that act as primers for DNA replication once primer is long enough, DNA polymerase can continue the process. primers are needed on both the leading and lagging strands; it is just not needed as much on leading strand because DNA polymerase can synthesize larger fragments at once.

how are RNA primers removed?

RNA primers are removed by 5' to 3' exonuclease activity and the gap is filled in by DNA polymerase (gap ~ 10-15 nucleotides long) and joined by DNA ligase. if removed from other side of primer, it would be endonuclease activity since it is already attached to DNA sequence. endonuclease = cleave within sequence exonuclease = cleave at the end of sequence with directionality 3' to 5' exonuclease = cleave at 3' end 5' to 3' exonuclease = cleave 5' end

what is the lifetime of an mRNA?

RNAs are very short lived, not protected as well as DNA in eukaryotes, mRNA half-lives vary from less than 30 minutes to 20 hours short lived mRNAs code for regulatory proteins, levels of which vary rapidly in response to environmental stimuli mRNAs encoding structural proteins or central metabolic enzymes have long half lives degradation of eukaryotic mRNA is initiated by shortening the poly-A tails (longer tail = longer half life), removing 5' cap or poly-A tail makes mRNA less protected and more likely degradable by nucleases

RNase (KEY EXP)

RNase was denatured, disulfide bonds broke. when put in solution again, spontaneously formed back into original conformation. it occurred very slowly though- far too slowly to operate alone within a cell. all information for correct conformation is provided by amino acid sequence.

Ex: GTP binding protein Ras

Ras = protein very important in signaling cell cycle, oncogene protein - when Ras is GTP bound, it is active and initiates signaling cascade to push cell cycle forward. - subtle conformational differences between inactive GDP bound and active GTP bound forms. - small difference in conformation determines whether Ras can interact with its target molecule, which signals the cell to divide. - about 25% of human cancers have mutated Ras, which locks it in the active GTP-bound conformation, continually signaling cell division

DNA damage induced by radiation and chemicals:

UV light induces the formation of pyrimidine dimers (if two pyrimidines are sitting next to each other on DNA, can form rings/dimers/bonds). alkylation of guanine residues (ethyl group added) reaction with carcinogens (smoking, etc.) can lead to addition of bulky groups addition of ethyl or methyl groups to various positions of DNA bases (alkylating agents are reactive compounds that can transfer methyl or ethyl groups to a DNA base) *this damage can block replication or transcription and lead to a high frequency of mutations*

Repetitive sequences

a large portion of complex eukaryotic genomes consists of highly repeated DNA sequences, which can be present in hundreds of thousands of copies per genome.

gene

a segment of DNA that is expressed to yield a functional product, which may be either an RNA (rRNA or tRNA) or a polypeptide

gene family

a set of several similar genes with similar biochemical functions, which formed by duplication of a single ancestral gene, followed by mutation and divergence.

how much of the genome do transposons take up?

about 50%

DNA repair

accurate transmission of genetic information is essential and requires that the parental cell accurately replicates its genome. DNA polymerase is not a perfect enzyme, repair mechanisms are in place. 3 billion bp sequence must be replicated 50 trillion times to produce one human-- need repair mechanisms. mutation rates indicate that error frequency during replication is less than one incorrect base per 10^9 nucleotides, which is much lower than predicted simply on basis of complimentary base pairing.

acetylation of lysine residues of histone proteins:

acetyl groups are added to lysine by histone acetyltransferase (HAT) and removed by histone deacetylase (HDAC); transcriptional activators and repressors are associated with HAT and HDAC lysine acetylation is reversible. process can be reversed as soon as transcription is finished. HATs are recruited by transcriptional activators = chromatin decondensation = transcriptional activation (HATs are coactivators) HDACs are recruited by transcriptional repressors = chromatin condensation = transcriptional repression (HDACs are coactivators)

glycosylation

adds carbohydrate chains to proteins to form glycoproteins carbohydrate moities play important roles in protein folding in the ER, in targeting proteins for transport, and as recognition sites in cell-cell interaction. starts in the ER before translation is complete N-linked glycoproteins = attached to N atom in side chain of asparagine O-linked glycoproteins = attached to O atom in side chain of serine or threonine

transfer RNA

align amino acids with corresponding codons on the mRNA template; they are 70-80 nucleotides long and have characteristic cloverleaf structures resulting from base pairing between different regions. 3' overhang = amino acid attachment site RNAPIII transcribes pre-tRNA transcript which undergoes a series of processing steps to generate mature tRNA sequences for tRNAs are not identical but they all have CCA sequence at 3' end where amino acids covalently attach to ribose of terminal adenosine anticodon loop binds to appropriate codon by complimentary base pairing

example: globin gene family

alpha and beta subunits of hemoglobin are both encoded by gene families in the human genome, with different members of these families being expressed in embryonic, fetal, and adult tissues. they are clustered on the same chromosomes (alpha globin locus on ch16 and beta globin locus on ch11). each family contains genes that are specificially expressed in embryonic, fetal, and adult tissues, in addition to nonfunctional gene copies (= pseudogenes) fetal globins have higher affinity for O2 than adult globins (carry more O2), allowing fetus to obtain O2 from maternal circulation

Protein misfolding diseases

alzheimer's disease, parkinson's disease, and type 2 diabetes are associated with aggregation of misfolded proteins. the misfolded proteins form fibrous aggregates called amyloids, characterized by beta sheet structures cystic fibrosis has improper folding of CFTR protein which transports Cl- ions across epithelial cell membranes. alzheimer's disease characterized by two aggregate types: - neurofibrillary tangles (misfolded tau proteins) - amyloid plaques (aggregates of misfolded amyloid beta protein)

is the amount of DNA in the intron sequences more or less than that in the exons?

amount of DNA in the intron sequences is often greater than that in the exons. protein-coding sequences account for only about 10% and introns comprise about 90% of the average human gene. introns are present in most eukaryotic genes but not all. almost all histone genes lack introns. clearly, introns are not required for gene function.

how is supercoiling of parental DNA ahead of replication fork relieved?

as DNA unwinds, the DNA ahead of the replication fork is forced to rotate which causes the DNA to twist and coil. topoisomerases = enzymes that catalyze the reversible breakage and rejoining of DNA strands two types: 1. topoisomerase 1 = break just one strand of DNA 2. topoisomerase 2 = introduce simultaneous breaks in both strands

3' polyadenlylation

at the 3' end, a poly-A tail is added by poly-A polymerase (AAAA...., bunch of adenines) in original DNA, there are poly-A sites that signal for poly-adenylation 1. upstream element 2. downstream GU rich element 3. specific sequence in between endonuclease cleavage between middle element and downstream element poly-A polymerase adds about 200 adenines to form poly-A tail in place of downstream element poly-A tail regulates translational rates and mRNA stability recognition of the poly-adenylation signal leads to termination of transcription, cleavage, and polyadenylation of the mRNA

Nonstandard codon-anticodon base pairing

base pairing at the third codon position is relaxed, allowing G to pair with U, and inosine in the anticodon to pair with U, C, or A. ex. phenylalanine base pairing - guanosine can pair with uridine (G=U), only two bonds inosine = unique nucleotide that can pair with U, C, or A nonstringent base pairing is important for redundancy in of genetic code. most amino acids are specified by more than one codon. many amino acids can be attached to more than one species of tRNA. some tRNAs can also recognize more than one codon in the mRNA.

consensus sequences

bases most frequently found in different promoters i.e. experiments show functional importance of -10 and -35 regions. - genes with promoters that differ from consensus sequences are transcribed less efficiently - mutations in these sequences affect promoter function - the sigma subunit binds to both regions

enhancer sequences

bind transcription factors that regulate transcription but function independent of proximity and orientation to TSS they are regulatory sequences located further away from TSS (can be more than 50,000bp away from promoters that they regulate). i.e. first identified in studies of the promoter virus sv40. found that activity of enhancers does not depend on distance from or orientation with respect to TSS. adding enhancer = more transcription.

promoter vs enhancer

both are cis-acting regulatory sequences that bind transcription factors. location of promoter DOES matter, transcription does not occur if move to different location. location of enhancer DOES NOT matter, has same effect no matter where it is (upstream, downstream, diff chromosome, even if flip sequence around backwards)

protein disulfide isomerase (PDI)

catalyzes disulfide bond formation. PDI is abundant in the ER where an oxidizing environment allows S-S linkages; for cysteine amino acids.

peptidyl prolyl isomerase

catalyzes isomerization of peptide bonds that involve proline residues (= cannot turn or twist well, very inflexible amino acids due to ring structure) isomerization between cis and trans configurations of prolyl peptide bonds could otherwise be a rate limiting step in protein folding want amino acids to be in trans configuration = favored

DNA polymerase

catalyzes phosphodiester bond formation by adding a deoxyribonucleoside 5' triphosphate to the 3' hydroxyl group of a growing DNA strand (primer strand) synthesis is always 5' -> 3' and DNA polymerase reads the DNA 3' -> 5' multiple DNA polymerases, they play different roles can only extend a pre-existing polynucleotide (primer), which must be bound to a template strand by complimentary base pairing.

rearrangement of nucleosomes via chromatin remodeling factors

chromatin remodeling factors = protein complexes that alter contacts between DNA and histones (repositioning, changing conformation, or ejecting) to allow for binding of transcriptional machinery

chaperones

class of proteins that facilitate folding of other proteins

proteolysis

cleavage of a polypeptide chain removes portions such as the initiator methionine from the amino terminus

chromatin

complexes between eukaryotic DNA and proteins, which typically contains about twice as much protein as DNA. major proteins of chromatin are histones basic structural unit = nucleosome

prokaryotic RNA polymerase

composed of 5 types of subunits sigma subunit is necessary for initiation. it identifies the correct sites for transcription initiation. most bacteria have several different sigma subunits that direct RNA polymerase to different start sites under different conditions.

how are the genomes of eukaryotes packaged?

composed of multiple chromosomes, each containing a linear molecule of DNA the DNA is tightly bound to histones (small basic proteins( that package the DNA in an orderly way in the cell nucleus.

nucleosome

consists of DNA wound around a protein core of 8 histone molecules (two of each H2A, H2B, H3, H4).

2 forms of spontaneous DNA damage

deamination = loss of amino groups in cytosine and adenine; cytosine becomes uracil and adenine becomes hypoxanthine depurination = loss of purine bases (adenine, guanine) and deoxyribose, leaving an apurinic (AP) site in DNA

autophagy

digestion of components from within the cell (i.e. dead mitochondria) activated in nutrient starvation, allowing cells to degrade nonessential proteins and organelles and reutilize the components plays a role in many developmental processes, such as insect metamorphosis, which involve extensive tissue remodeling.

DNA proofreading

during synthesis, DNA polymerase has a proofreading ability. DNA polymerases III (E. coli), epsilon, and delta (eukaryotes) exhibit 3' to 5' exonuclease activity. when an incorrect base is incorporated, it is removed by the exonuclease activity. DNA polymerase synthesizes in 5' to 3' direction which allows exonuclease activity in 3' to 5' direction because it can remove incorrect nucleotide. this is a reason why it is a slower enzyme.

Ex. cyclins that regulate progression through division cycle of eukaryotic cells

entry of cells into mitosis is controlled in part by cyclin B, a regulatory subunit of Cdk1 protein kinase. degradation of cyclin B by proteasome then leads to inactivation of cdk1 kinase, allowing the cell to exit mitosis and return to interphase. transition from metaphase to anaphase requires inactivation of cyclin B. cdk1 also trigger ubiquination of cyclin B, leading to its degradation by the proteasome.

aminoacyl-tRNA synthetase

enzyme that attaches amino acids to specific tRNAs about 20 different aminoacyl-tRNA synthetases that each recognize: - particular amino acid - cognate tRNAs to which the amino acid should be attached

O6-methylguanine methyltransferase

enzyme that recognizes the alkylation of guanine and fixes the mistake

eukaryotic promotor sequences

eukaryotic genes have promoters that are more complex, containing combinations of several core promoter elements i.e. TATA box sequence - resembles -10 of bacterial promoters

chromatin condensation

extent of chromatin condensation varies during the life cycle of the cell and plays an important role in regulating gene expression. in interphase (nondiving), most of chromatin is in euchromatin state = decondensed and distributed throughout the nucleus. in this phase, genes are transcribed and DNA is replicated. usually 30nm fibers. about 10% of interphase chromatin is in highly condensed state that resembles the chromatin of cells undergoing mitosis = heterochromatin = transcriptionally inactive and contains highly repeated DNA sequences

Ex. regulation of ferritin translation in response to iron levels (translational regulation of specific mRNAs)

ferritin = protein that stores iron ferritin is synthesized to store iron, only needed when iron is present. adequate iron = initiation factors recognize IRE and small subunit binds to begin scanning for translation = ferritin protein produced iron scarce = iron regulatory protein (IRP) binds to IRE in 5' UTR and blocks translation (saves cell energy)

tertiary structure

folding of the entire polypeptide into its functional 3D structure. results from interactions between side chains of amino acids.

how is RNAPII recruited to promoters?

general transcription factors = proteins involved in transcription from polymerase II promoters

pseudogenes

genes that are not functional but look like normal gene; inactivated by mutations. get transcribed almost as often as protein-coding but do not have functional significance.

eukaryotic genomes vs. prokaryotic genomes

genomes of most eukaryotes are larger and more complex than those of prokaryotes. difference in sizes of genomes primarily reflects differences in amounts of noncoding DNA, rather than differences in the numbers of protein-coding genes. genome size or # protein coding genes is not related to genetic complexity

how is parental double stranded DNA separated and stabilized?

helicase = enzyme that catalyzes the unwinding of parental DNA ahead of the replication fork. requires ATP. single stranded DNA binding proteins (SSB) = stabilize the unwound template DNA so that it can be copied by the polymerase (keep it single stranded, prevent from clumping up)

relationship between transposons and mutations

highly repetitive SINEs and LINEs are not useful for the cell in which they are located. transposons induce mutations when they integrate at a new target site and are usually harmful. mutations resulting from transposition of SINEs and LINEs have been associated with several inherited human diseases (hemophilia, CF, MD, hereditary cancers)

ex. immunoglobulin enhancer

immunoglobulin gene = helps make antibodies this enhancer is active in lymphocytes but not in other cell types; this regulatory sequence is partly responsible for tissue specific expression of immunoglobulin genes ~200 bp with multiple sequence elements that bind different regulatory proteins that work together to regulate gene expression mutation of any one of the enhancer binding sites reduces transcription but does not block all together transcription still occurs even if mutate all enhancer sequences

why can't DNA be synthesized in the 3' to 5' direction?

in DNA replication, energy in adding a nucleotide comes from the phosphate groups.... lose 2 phosphates to form phosphodiester bond (need energy) if DNA synthesis proceeded 3' to 5', then excision of a terminal 5' nucleotide would prevent replacement of a new nucleotide. 3 phosphates are needed to make the bond and removal of the 5' nucleotide leaves only one. the 3' hydroxyl group is there but only a single phosphate on the 5'. dehydration to form bond is not possible.

methylation mechanism in prokaryotes

in bacteria, adenines are methylated in specific sequences ~4,000 bp. MutH uses the methyl group as a guide on where to cut. Methyl groups are on parental strand. newly synthesized DNA is not yet modified by methylation allowing recognition of new vs. parental strands. this is specific to prokaryotes which is why MutH is only present in mismatch repair for prokaryotes (E. coli)

origins of replication in humans

in humans, there are multiple origins of replication on each chromosome. this is necessary in order to repliate the size of eukaryotic genomes in a timely manner. eventually the DNA polymerases meet up (synthesizing in both direction of multiple ori and replication forks) and fall off. fragments are then joined by DNA ligase.

when does splicing take place?

it occurs as transcription takes place. association of splicing factors with CTD assures that the exons are joined in 5' to 3' order. splicing factors = proteins that facilitate splicing by snRNPs by guiding them to correct splice sites

upstream regulatory sequences

located close to TSS about 50-1000bp upstream, bind additional proteins/trans-acting factors that regulate transcription - about 10% genes encode transcription factors (trans-acting) - different trans-acting factors will recognize these specific sequences, bind them, and initiate transcription. only about 5-6 nucleotides are needed for protein to recognize and bind. - transcription factors act as either activators or repressors

mRNA localization

mRNA can be localized to specific subcellular locations which controls where the protein is made. proteins that bind to 3' UTRs are also responsible for localizing mRNAs to specific regions of cells. the 3'UTR of mRNAs destined for subcellular localization contain specific sequences that target them for transport. they are bound by mRNA localization proteins that transport them to their correct destination. EXP: localization of mRNA in Xenopus oocytes (in situ hybridization) - blue labeled Xlerk mRNA generated - injected into Xenopus oocytes - examined where or if localized - found that they localized to vegetal pole

how were cis-acting regulatory sequences in eukaryotic genes identified?

many cis-acting DNA sequences regulate expression of eukaryotic genes. these regulatory sequences have been identified by gene transfer assays (usually upstream of TSS) - regulatory sequences are ligated to reporter genes that encode easily detectable enzymes - regulatory sequence directs expression of reporter gene in cultured cells (measurable output) - regulatory sequence drives transcription of gene

protein-protein interactions

many proteins consist of multiple subunits. interactions between them can regulate protein activity.

photoreactivation

mechanism of DNA repair in which visible light breaks the UV-induced pyrimidine dimer. many types of cells use photoreactivation but it is not universal. humans lack this mechanism of repair because do not have photoreactivating enzyme that gets energy from the sun.

microRNAs (miRNAs) = type of noncoding RNA

miRNA genes are transcribed to yield primary transcribed that contain hair pin structures. Primary miRNAs are sequentially cleaved by the nucleases Drosha and Dicer to yield ds miRNAs, which associate with the RISC complex in which the two strands of the miRNA are unwound. the miRNA then targets RISC to the 3' UTR of a target mRNA, leading to repression of translation and degradation of mRNA.

Mut S and MutL mutations, colon cancer

mutations in the human homologs of MutS and MutL cause inherited colon cancer, one of most common inherited diseases. doesn't appear immedietly ... mutation itself does not cause cancer, there needs to be a mistake that ultimately doesn't get fixed (b/c mutation in MutS and MutL); if mistake occurs in cancer gene then, cancer could result mutations in MutS and MutL increase susceptibility to the colon cancers can acquire mutations in genes that do regulate cell cycle but never get fixed because lack of repair mechanism or improper function of repair mechanism (MutS and MutL) defects in these genes result in a high frequency of mutations in other cell genes, and the likelihood that some will eventually lead to development of cancer

negative and positive transcriptional control

negative control = mechanisms that inhibit transcription positive control = mechanisms that activate transcription

Are introns present in prokaryotes?

no, introns are rarely seen in prokaryotes. E. coli genome is 88% protein-coding sequences. much more efficient in using its DNA than in humans.

can RNA polymerase distinguish between introns and exons?

no. the entire gene is transcribed and the introns, which are non protein coding, are removed during post-transcriptional splicing.

introns

noncoding DNA sequences location WITHIN genes that are transcribed along with the coding sequences (exons). the coding sequences of gene (exons) are separated by the introns. removed from the mRNA post-transcriptionally in splicing process. introns account for about 35% of the human genome

long noncoding RNAs (lncRNAs)

noncoding RNAs greater than 200 nucleotides long that have recently become recognized as major regulators of gene expression in eukaryotic cells. i.e. involved in X chromosome inactivation.

what role do noncoding sequences play?

noncoding sequences play roles in regulation of gene expression and expanding coding potential by allowing genes to be expressed in alternate ways

triplet codons

nucleotide sequence is read in triplet codons, each of which is responsible for a specific amino acid. codons are read 5' to 3' to direct polypeptide synthesis amino (N) to carboxyl (C) terminus; (*transcription and replication DNA is read in 3' to 5' direction*) AUG = start codon UAA, UAG, UGA = stop codons 5' and 3' ends of mRNA are not translated into protein (called 5' and 3' UTR)

ori in E. coli

ori is a single 245 bp DNA sequence one key characteristic of a plasmid vector is that it can replicate independently in bacteria and produce large quantities of DNA insert due to origin of replication

Ex. origins of replication in S. cerevisiae

origins of replication of eukaryotic chromosomes were first studied in yeast, S. cerevisiae. they were identified as sequences that could support the replication of plasmids in transformed cells. experiment: both plasmids I and II contained a selectable marker gene. only plasmid II contained origin of replication (ARS = autonomously replicating sequence). plasmid I obtained only rare transformants in which plasmid has been integrated into chromosomal DNA. plasmid II was able to replicate without any integration into a yeast chromosome (autonomous replication) so many more transformants resulted.

protein folding

polypeptide chains must undergo folding and other modifications to become functional proteins

RNA polymerase

principle enzyme for RNA synthesis (transcription) synthesizes 5' to 3', reads the DNA 3' to 5'

mediator

protein complex of 20+ subunits that interacts with both general transcription factors and RNA polymerase

protein degradation

protein levels in cells are determined by rates of synthesis and rates of degradation. half lives of proteins vary greatly. many regulatory proteins have short half lives allowing levels to change quickly in response to external stimuli. faulty or damaged proteins are recognized and rapidly degraded differential rates of degradation are important in cell regulation

two classes of kinases:

protein-serine/threonine kinase protein-tyrosine kinase

trans-acting factors

proteins that regulate a gene's expression and are encoded by different gene elsewhere in the genome (i.e. repressor)

promoter

region of DNA upstream of transcription start site (TSS) where RNA polymerase binds to initiate transcription of a gene. contains specific DNA sequences that recruit RNA polymerase.

retroviruses and retrotransposons

retroviruses contain RNA genomes in their virus particles but replicate via the synthesis of a DNA provirus in infected cells. provirus is synthesized by reverse transcriptase and integrated into the chromosomal DNA by integrase. some retrotransposons are structurally similar to retroviruses. retrovirus like elements encode reverse transcriptase and integrate and can move to new chromosomal sites within the same cell but cannot spread from one cell to another through infectious particle packaging. LINEs encode reverse transcriptase and an enzyme for integration. SINEs do not encode reverse transcriptase or other proteins.

telomeres

sequences at the ends of eukaryotic chromosomes that play critical roles in chromosome replication and maintenance consist of repeats of simple sequence DNA containing clustered of G residues on one strand. telomere repeat in humans is TTAGGG. they are repeated hundreds of times and terminate with a 3' overhand of ssDNA. form loops at the end of chromosomes because all the sequences are simple sequence repeats so complimentary to eachother, hybridizes with complimentary strand. bind a protein complex (shelterin) that protects the chromosome from degradation.

mammalian genome size:

sequences of ~3 billion base pairs of DNA only ~1.2% protein-coding sequence, 98.8% does not code for proteins. about 20,000-25,000 genes

which amino acids can be phosphorylated?

serine, threonine, tyrosine

centromere

sites where sister chromatids attach, largely composed of simple-sequence repeats attachment site for spindle fibers that attach at kinetochore (protein complex) centromeres are vastly different between species centromeres in humans mostly made of satellite DNA (simple sequence repeats)

lysosomal proteolysis

slow, gradual turnover of proteins and other cell constituents protein degradation can take place in lysosomes = membrane enclose organelles that contain digestive enzymes, including proteases lysosomes digest extracellular proteins taken up by endocytosis, and take part in turnover of organelles and proteins, can degrade just about anything

histones

small proteins containing a high proportion of basic amino acids (arginine and lysine) that facilitate binding to the neg. charged DNA molecule. lysine and arginine are pos. charged amino acids and DNA is neg. charged. the pos. charge allowed the histone proteins to do their job, facilitates interaction with the DNA.

ARS element

span about 100 bp including an 11 bp core sequence common to many different ARS (ACS consensus sequence) consensus sequence is essential for ARS function and has been found to be the binding of ORC = origin recognition complex. ORC is required for initiation of DNA replication because it recruits other proteins (helicase, etc.)

how is specificity of enhancers maintained?

specificity is maintained partly by insulators, which divide chromosomes into independent domains and prevent enhancers from acting on promoters located in an adjacent domain. insulators give DNA a 3D structure that holds it in place genomes are divided into discrete chromosomal domains = topologically associating domains (TADs) enhancers and promoters within a TAD interact frequently with each-other but only rarely with elements in other domains. main protein that binds insulators in vertebrates is CTCF TADs allow for enhancer sequences to always be accessible and close to TSS. regions of DNA being held together on purpose for regulation of gene expression.

splicing

splicing machinery = spliceosome = small nuclear ribonucleoprotein particles (RNA and protein working together, snRNPs), have 5 types of small nuclear RNAs - U1, U2, U4, U5, U6 - that are connected to 6-10 protein molecules to form snRNPs slicing proceeds in two steps: 1. cleavage at 5' slice site (SS) and joining of the 5' end of the intron to an adenine within the intron (branch point). the intron forms a loop between 5' SS and branch point. 2. cleavage at the 3' SS and simultaneous ligation of the exons excises the intron loop

linker DNA

stretch of DNA separating two nucleosomes

Transcription

synthesis of an RNA molecule from a DNA template specialized cells have the same # genes, they are different because they express the genes differently = only a subset of genes are actually expressed (i.e. telomerase expression is higher in germ and embryonic cells than adult somatic; i.e. many genes encoding DNA replication machinery are expressed during replication but not during G1, G2, and M phases

translation

synthesis of proteins as directed by mRNA template, first step in formation of functional proteins translation occurs in the cytosol, transported out of nucleus translation is carried our on ribosomes with tRNAs serving as adaptors between codons and amino acids

Ubiquination

targets a protein for degradation by a proteasome. 3 enzymes involved: E1 = Ub-activating enzyme (ATP dependent) E2 = Ub-conjugating enzyme (brings to E3 molecule) E3 = Ub-ligase enzyme (ligates ubiquitin to target protein) amount of E3>E2>E1 in cell.

Telomeres and Telomerase

telomeres are the terminal sequences of linear DNA molecules which are tandem repeats of simple sequence DNA (sequence differs depending on organism) telomeres are maintained by telomerase = enzyme that catalyzes synthesis of telomeres in the absence of a DNA template two key features of telomerase: 1. specialized reverse transcriptase = make DNA from RNA 9class of DNA polymerases) 2. carries its own template RNA which is complimentary to the telomere repeat sequences the RNA template allows telomerase to extend the 3' end of template DNA by one unit beyond its original length. the complimentary sequence can then be synthesized by the polymerase alpha-primase complex.

synthesis of leading and lagging strands of DNA

the leading strand is synthesized continuously in the direction of replication fork movement. the lagging strand is synthesized in small pieces (Okazaki fragments) backward from the overall direction of replication fork. this is discontinuous replication. the okazaki fragments are then joined by DNA ligase

what is one reason for slower DNA replication in eukaryotes?

the presence of histones, wrapping up the DNA tightly

allosteric regulation

the regulation of enzymes by small molecules that bind to a site distinct from the active site, changing the conformation and catalytic activity of the enzyme. binding of small molecule can be inhibitory or activating. allosteric site binding changes conformation of protein and its active site (where substrate binds). this conformational change either inhibits binding of substrate or activates binding of substrate. i.e. eIF2 is an example of a protein regulated by small molecules. it is subject to allosteric regulation by GTP/GDP binding. i.e. repressor protein in lac operon is under allosteric regulation. binding of lactose changes its conformation and prevents it from binding to the mRNA. i.e. feedback inhibition. molecule starts by binding to active site in one protein and changes, then binds another protein and changes, continually repeats down pathway until final product formed. final product can bind to initial protein in allosteric site, inhibiting the pathway.

global regulation of translation

translation can be regulated by modification of initiation factors, resulting in global effects on translational activity. eIF2 in high energy GTP-bound state carries the methionine charged tRNA. cells need to regenerate eIF2-GTP in order for it to keep binding to charged tRNA. eIF2B exchanges GDP or GTP on eIF2. this cycle continues when cells are healthy and in presence of growth factors. when the cell is under stressful conditions or there are no growth factors, eIF2 and eIF2B are inhibited by phosphorylation. regulatory protein kinases block exchange of bound GDP or GTP, inhibiting translation initiation. eIF4E (binds 5' cap) is regulated by protein-protein interactions with 4E binding protein (4E-BP). in absence of growth factors, the nonphosphorylated 4E-BP binds to eIF4E and inhibits translation. growth factors activate protein kinases that phosphorylate regulatory proteins (4E-BP). 4E-BP does not bind eIF4E and normal initiation complex is formed for translation to occur.

ribosomes

translational machinery consist of a large subunit and small subunit; each subunit is composed of multiple rRNAs and polypeptides 4 different rRNAs: 5S, 28S, 18S, 5.8S assembly of all rRNAs into subunits of ribosomes happen in nucleolus rRNAs within the large subunit catalyze peptide bond formation; ribosomes build polypeptides one amino acid at a time

what is the role of TFIIH?

two roles: 1. helicase activity = separate DNA 2. CTD kinase = phophorylates the C terminal domain (necessary to initiate transcription; if not phosphorylated, transcription does not occur)