PCB 3134 EXAM 4 REVIEW

Transcription in prokaryotes. What components an E.coli promoter has?

- A single type of RNA polymerase composed of 5 subunits that are tightly associated to form a core enzyme. -An accessory polypeptide, sigma factor (s), increases the enzyme's affinity for promoter sites in DNA. The polymerase, s factor, and DNA with the strands separated is called the Open Complex. -Once about 10 nucleotides have been incorporated, the enzyme undergoes a conformational change into a transcriptional elongation complex that can move processively. --------The basic elements of a promoter region in the DNA of E. coli +1 nucleotide: at which transcription is initiated -1 nucleotide: preceding the +1 nucleotide Upstream: those portions of DNA preceding the initiation site. Downstream: those portions of DNA succeeding the initiation site. Consensus sequence: present in the promoters of many genes. -35 element: the TTGACA consensus sequence around -35 site. Pribnow box (-10 element): the TATAAT consensus sequence around -10 site.

Centromere.

- Centromere is a site where the outer surfaces are markedly indented. 1. Centromere contains tandemly repeated sequences, which vary from one species to another species; 2. Centromeric DNA associates with specific proteins, such as protein that serve as attached sites (kinetochores) for the microtubules that separate chromosomes during cell division.

What is karyotype?

- Chromosomes are cut out of a photograph of metaphase chromosomes, matched up into homologous pairs, and ordered according to decreasing size, forming a karyotype.

DNA replication direction: 5' to 3'. DNA replication requires a template and primer (provides 3' OH).

- DNA polymerase 1. A template DNA strand to copy; 2. A primer to which nucleotides can be added (Primer is a DNA strand provides the necessary 3' OH terminus). - DNA polymerase can only synthesize DNA in a 5'-to-3' (5'3') direction.

What is "the end-replication problem" and how is it solved?

- DNA polymerases don not initiate the synthesis of a strand of DNA, but only add nt to the 3' end of an existing strand.

How does meiosis increase the genetic variability in a population of organisms from one generation to the next?

- Meiosis increases the genetic variability in a population of organisms from one generation to the next by: 1. Independent assortment of maternal and paternal chromosomes. 2. Genetic recombination (crossing-over).

Replication origin, bidirectional replication, and replication fork.

- Origin: a specific site (oriC in E. coli) where a number of proteins bind to initiate the process of replication. -Bidirectional replication: once initiated, replication proceeds outward from the origin in both directions. - Replication forks: the sites where the pair of replicated segments come together and join the nonreplicated segments.

DNA replication is semiconservative. What is semiconservative replication and how to demonstrate it?

- Replication occurred by gradual separation of the strands of the double helix. Each strand can act as a template to direct the synthesis of the complementary strand and restore the double-stranded state. -As a result, each of the daughter duplexes consist of one complete strand inherited from the parental duplex and one complete strand that has been newly synthesized. - Meselson and Stahl experiments: supported the semiconservative model of replication in bacterial cells.

Leading strand and lagging strand. How are they replicated? What is Okazaki fragment?

- Semidiscontinuous replication: one strand is synthesized continuously and the other discontinuously. -- Leading strand: the strand that is synthesized continuously. -- Lagging strand: the strand that is synthesized discontinuously. - Okazaki fragments: the small segments (1000-2000 nt in bacteria, ~150 nt in eukaryotes) synthesized during DNA replication.

Telomerase structure and function.

- Telomerase is a reverse transcriptase that uses a RNA molecule inside the enzyme as a template to add new repeat unit to the 3' end of the overhanging strand. -Functions: 1. complete the replication of chromosomes; 2. form caps that protect the chromosomes from nucleases and other destabilizing factors; 3. preventing the ends of chromosomes from fusing with one another.

4. Histone modification and histone code.

- The AA residues in the core histone tails are subject to modification, mainly methylation, acetylation, or phosphorylation. - Histone code is the pattern (combinations) of modifications which contains encoded information governing the properties of the chromatin. 1. The degree of compaction; 2. If a gene or cluster of genes will be transcribed or not.

Telomere structure and function.

- The tips of each chromosome DNA molecule consist of an unusual stretch of repeated sequences, which forms a cap at the end of the chromosome. The stretch of repeated sequences is a telomere. - The human telomeres contain the sequence (TTAGGG) which is the same throughout the vertebrates, and similar sequences are found in most other organisms.

What is treadmill? When does it happen?

-"Treadmill" of the microtubules of the metaphase spindle ---A net addition of subunits at the plus end ands a net loss of subunits at the minus ends cause a pole-ward flux of tubulin subunits in a mitotic spindle.

How is DNA synthesis initiated? Primase: a RNA polymerase.

---- The initiation of Okazaki fragments is accomplished by a distinct type of RNA polymerase (primase) that constructs a short RNA primer. ------A short RNA primer is also synthesized at the 5' end of the leading strand by primase. - The RNA primers are subsequently removed, and the resulting gaps are filled with DNA and then sealed by DNA ligase.

Structure of mRNA, 5' caps, 3' poly(A), and their functions.

-5' caps (methylguanosine caps) 1. The last of the three phosphates is removed, converting the 5' terminus to a diphosphate. 2. A GMP is added in an inverted orientation so that the 5' end of the guanosine is facing the 5' end of the RNA chain. The first two nucleosides are joined by an unusual 5'-5' triphosphate bridge. 3. The terminal, inverted guanosine is methylated at the 7 position on its guanine base, while the nucleotide on the internal side of the triphosphate bridge is methylated at the 2' position of the ribose. -The modifications at the 5' end occur while the RNA is still in very early stages of synthesis. -Functions of the 5' methylguanosine caps 1. Prevent the 5' end of the mRNA from being digested by exonucleases. 2. Aid in transport of the mRNA out of the nucleus. 3. Play an important role in the initiation of mRNA translation. -The poly(A) tail begins about 20 nt downstream from the sequence AAUAAA in the primary transcipt, which serves as a recognition site for a complex that carry out the processing reactions at the 3' end. 1. An endonuclease in the processing complex cleaves the pre-mRNA downstream from the recognition site. 2. An poly(A) polymerase adds 250 or so adenosines without the need of a template. -The poly(A) tail together with an associated protein protects the mRNA from premature degradation by exonucleases.

Which molecular motors are involved in mitotic movement and how?

-All of the motors involved in mitotic movements are microtubule motors, including a number of different kinesin-related proteins and cytoplasmic dynein. -Motor proteins located along the polar microtubules: keeping the poles apart and elongating the spindle during anaphase B. -Motor proteins residing on the chromosomal microtubules: moving the chromosomes during prometaphase, maintaining the chromosomes at the metaphase plate, and separating chromosomes during anaphase.

Understand the relationship between transcription and translation in prokaryotes and eukaryotes (see Figure 11.51).

-An E. coli chromosome engaged in transcription and translation -Cells of silk gland transcription in the nucleus translation in the cytoplasm

Polyribosome (polysome).

-As soon as each ribosome moves a sufficient distance along the mRNA from the initiation codon, another ribosome attaches to the mRNA and begins its translation activity. A complex in which a number of ribosomes are attached to one mRNA molecule is a polyribosome. -The simultaneous translation of the same mRNA by numerous ribosomes greatly increases the rate of protein synthesis within the cell.

3. Chromatin, euchromatin, and heterochromatin.

-Chromatin: euchromatin and heterochromation - Chromatin: the substance of chromosome, including DNA, chromosomal proteins, and chromosomal RNA - Euchromatin: chromatin that returns to dispersed state after mitosis. Euchromatin is relatively extended and open and at least potentially active. - Heterochromatin: chromatin that remains compacted during interphase, ~10% of the total chromatin. Heterochromatin is very condensed and its DNA is inaccessible. ----- Constitutive heterochromatin remains in the compacted state in all cells at all times, the DNA inside is therefore permanently silenced. Centromeres. ----- Facultative heterochromatin is specifically inactivated during certain phases of an organism's life or in certain types of differentiated cells. One of the X chromosomes of female cells.

Know the difference between mitosis and meiosis.

-Chromosome behavior: ------Mitosis: Homologous chromosomes independent -----Meiosis: Homologous chromosomes pair forming bivalents until anaphase I -Chromosome number- reduction in meiosis: -----Meiosis- daughter cells haploid -----Mitosis- identical daughter cells -Genetic identity of progeny -----Mitosis: identical daughter cells -----Meiosis: daughter cells have new assortment of parental chromosomes -----Meiosis: chromatids not identical, crossing over

What is the difference between animal cell cytokinesis and plant cell cytokinesis?

-Cytokinesis: a process dividing a cell into two daughter cells. 1. During anaphase an indentation of the cell surface in a narrow band is formed around the cell. 2. The indentation deepens to form a furrow that completely encircles the cell. The plane of the furrow lies in the same plane as the metaphase plate 3. Additional PM is delivered to the cell surface via cytoplasmic vesicles that fuse with the advancing cleavage furrow. 4. The furrow continues to deepen and opposite surfaces fuse in the center of the cell, splitting the cell in two. -Cytokinesis in plant cells -----Plant cells start cytokinesis by forming a partition called the cell plate in the center of the cell. The position of the cell plate is determined by the preprophase band formed in late G2. 1. During interphase, MTs are distributed throughout the cortex 2. As cell approaches mitosis, the MTs disappear from most of the cortex, leaving a single transverse preprophase band; 3. As the cell progress into mitosis, the preprophase band is lost and MTs reappear in the form of mitotic spindle; 4. After the chromosomes have been separated, the mitotic spindle disappear and is replaced by a bundle of MTs, the phragmoplast.

Components for transcription. Transcription direction and why the polymerization is irreversible? Which strand of DNA is used for transcription template?

-DNA-dependent RNA polymerases (RNA polymerases): the enzymes responsible for transcription in both prokaryotic and eukaryotic cells. -Template: the DNA strand whose sequence is complementary to the RNA molecule synthesized from it. -Promoter: the site on DNA to which an RNA polymerase molecule binds prior to initiating transcription. -Transcription factors: the proteins that are required to help RNA polymerases to recognize promoters. -Chain elongation: attack by the 3' OH of the nucleotide at the growing strand end on the 5'-P of the incoming nucleoside triphosphate -EM: several RNA polymerase molecules bound to a phage DNA template

How are rDNA genes arranged in the nucleoli? What is included in an rRNA transcription unit?

-During the growth of amphibian oocytes, the amount of rDNA in the cell is greatly increased as are the number of nucleoli. -A large number of small, nucleolar RNAs (snoRNAs) are packaged with particular proteins to form particles (snoRNPs, small, nucleolar ribonucleoproteins), which begin to associate with the rRNA precursors before it is fully transcribed. -snoRNPs function in processing of pre-rRNAs, such as removal of the 5' end of the transcript and cleavages of the pre-RNAs. 1. The tandem arrangement of the repeated rDNA genes. 2. Multiple (about 100) rRNAs are transcribed at the same time. 3. Besides RNA polymerase at the base of each fibril, the RNA fibrils contains clumps and associated particles (RNA and protein) that work together to convert the pre-rRNAs to their final rRNAs and assemble them into ribosomal subunits. 4. Nontranscribed spacer.

What are replication foci?

-In eukaryotes, replication forks that are active at a given time are not distributed randomly throughout the cell nucleus, but instead are localized within 50 to 250 sites, called replication foci.

How many stages are in a cell cycle? How are these stages defined?

-Interphase: the period between cell divisions, which is a time when the cell grows and engages in diverse metabolic activities. 1. G1: the period following mitosis and preceding DNA synthesis. -Cell grows and carries out normal metabolism; organelles duplicate. 2. S phase: the period in which DNA is replicated and additional histones are also synthesized. -DNA replication and chromosome duplication. 3. G2: the period between the end of DNA synthesis and beginning of M phase. -Cell grows and prepares for mitosis.

Remember stages in meiosis. Fives stages of prophase I and events in each stage of prophase I.

-Meiosis I ---Prophase I: leptotene, zygotene, pachytene, diplotene, diokinesis - Leptotene 1. Chromosomes begin to condense. 2. Near the end of leptotene, telomeres become localized at the inner surface of the nuclear envelope at one side of the nucleus. - Zygotene 1. Chromosomes continue to condense and shorten 2. Homologues associate with one another. This process of chromosome pairing is called synapsis. -- The synaptonemal complex (SC), a ladder-like structure, connects homologue chromosomes together. --Two lateral elements (mainly cohesin) --Transverse protein filaments -- Bivalent or tetrad - Pachytene ----The end of synapsis marks the beginning of pachytene, which is characterized by a fully formed synaptonemal complex. ----Within the synaptonemal complex there are protein complexes, called recombination nodules, that contain the enzymes required to complete the genetic recombination. ----Genetic recombination is completed by the end of pachytene. - Diplotene ------ Dissolution of the synaptonemal complex leaves the chromosomes attached to one another at a specific points by X-shaped structures, chiasmata (chiasma). - Diakinesis 1. The chiasmata move to the ends of the chromosomes in a process called terminalization. 2. Meiotic spindle is assembled. 3. Disappearance of nucleolus and breakdown of nuclear envelope. 4. The tetrads (bivalents) move to the metaphase plate. ---Metaphase I ---Anaphase I ---Telophase I -Interkinesis -Meiosis II ---- Prophase II ---Metaphase II ---Anaphase II ---Telophase II

How many different kinds of RNAs a cell has (remember there are other RNAs besides the three groups we covered in the class)? What is their function?

-Messenger RNAs (mRNAs): RNAs that include nucleotide code information for polypeptide synthesis. -Ribosomal RNAs (rRNAs): RNA components of ribosomes. -Transfer RNAs (tRNAs): RNAs that translate the information in the mRNA nucleotide code into AA order of a polypeptide. -Other RNAs: small nuclear RNAs (snRNAs) small nucleolar RNAs (snoRNAs) small interfering RNAs (siRNAs) microRNAs (miRNAs) -Many RNAs fold into a complex three-dimensional shape, which is markedly different from one type of RNA to another. RNA folding is driven by the formation of regions having complementary pairs. -RNAs often contain nonstandard base pairs and modified nitrogenous bases. the unorthodox regions serve as recognition sites for proteins and other RNAs, promote RNA folding, and help stabilize the structure of the molecule.

Metaphase plate, astral microtubules, chromosomal microtubules, and polar microtubules.

-Metaphase: Alignment of chromosomes to the center of the mitotic spindle marks the start of metaphase --Metaphase plate: in the middle of the cell --Astral microtubules --Chromosomal (or kinetochore) microtubules --Polar (or interpolar) microtubules

Definition of mitosis and cytokinesis.

-Mitosis: a process of nuclear division in which the replicated molecules of each chromosome are faithfully partitioned into two nuclei. -Cytokinesis: a process by which a dividing cell splits in two, partitioning the cytoplasm into two cellular packages.

What are the five stages of mitosis? How are they defined? Remember events in each stage.

-Mitosis: prophase, prometaphase, metaphase, anaphase, telophase -Prophase: 1. Formation of the mitotic chromosome -Chromosome compaction (condensation) -Chromosome scaffold -Condensin -Cohensin 2. Formation of the mitotic spindle 3. The dissolution of the nuclear envelope -Prometaphase --The dissolution of the nuclear envelope marks the start of prometaphase. 1. The microtubules of the spindle penetrate into the central region of the cell; 2. The free ends of the microtubules grow and shrink in a highly dynamic fashion, searching for chromosomes; 3. The two sister chromatids become connected by their kinetochores to microtubules that extend from opposite poles; 4. The chromosomes are moved by a process called congression toward the center of the mitotic spindle. -Metaphase: Alignment of chromosomes to the center of the mitotic spindle marks the start of metaphase. -Anaphase: the sister chromatins of each chromosome split apart and start their movement toward opposite poles. 1. Anaphase promoting complex (APC) is activated by association with Cdc20 that is synthesized late in the cell cycle; 2. Activated APC ubiquitinates a major anaphase inhibitor securin that secures the attachment between sister chromatids; 3. Ubiquitination and degradation of securin at the end of metaphase releases an active protease separase that cleaves a key subunit of the cohesion molecules, triggering the separation of sister chromatids. -Telophase ---As the chromosomes reach their respective poles, they tend to collect in a mass, which marks the beginning of telophase. ---Chromosomes become dispersed; -Nuclear envelope reforms; -Golgi complex and ER reform.

Specific transcription factors and their functions.

-Once transcription begins, certain of the GTFs (including TFIID) may be left behind, while other are released from the complex. The TFIID can initiate additional rounds of transcription. -A variety of specific transcription factors are required to bind at numerous sites in the regulatory regions of the promoter DNA to determine: 1. whether or not a preinitiation complex assembles at a promoter; 2. the rate at which the polymerase initiates new rounds of transcription from that promoter. -Termination of transcription by RNA polymerase II is not well understood; a transcribing RNA polymerase II can travel a variable and extensive distance past the point that will ultimately give rise to the 3' terminus of the transcribed mRNA.

How is genomic DNA packaged into chromosomes? The ratio of each packing level.

-Packaging genomic DNA into chromosomes: Nucleosome filament (10 nm): Packing ratio: ~7:1 30 nm fiber: Packing ratio: 6:1 (~40:1 in total) 80-100 nm fiber Chromosome Nucleosome: core histones and their combinations inside a nucleosome. - How was it found that the first level of chromosome organization is nucleosomes? ----In early 1970s, it was found that chromatin treated with nonspecific nucleases resulted in fragments of 200 bp in length, whereas similar treatment of naked DNA produced a random sized population. ---- In 1974, Roger Kornberg (Harvard University) proposed that DNA and histone proteins are organized into repeated subunits, called nucleosomes. -How is a nucleosome assembled? Where is the H1 histone protein located? --- Nucelosome (core nucleosome, nucleosome core particle): 146 bp of supercoiled DNA wrapped almost twice around a histone octamer containing all four core histones (H2A, H2B, H3, and H4). --- The eight histone molecules are organized into four heterodimers: two H2A-H2B and two H3-H4 dimers. ---- H1 histone (linker histone) binds to part of the linker DNA that connect one nucleosome core particle to the next. ---- H1 histone and the histone octamer interact with about 168 bp of DNA ---- H1 histone molecules can be selectively removed from the chromatin fibers by subjecting the preparation to solutions of low ionic strength. --- Chromatin fibers released from the nucleus of a Drosophila cell in a buffer of low ionic strength. - What are the characteristics of histones? ---- Histones are a group of proteins that possess an unusual high content of the basic AAs arginine and lysine. ---- Histones are divided into 5 classes which can be distinguished by their Arg/Lys ratio. ---- The AA sequences of histones, especially H3 and H4, are very conserved. However, there are several alternate versions of the core histones in most cells, which may have specialized functions. - How are DNA and histones are held together? ----- DNA and core histones are held together by noncovalent bonds, including ionic bonds between negatively charged phosphates of the DNA backbone and positively charged residues of the histones. - How can the 30 nm fiber of chromatin be resolved? Which proteins may play a function in formation of this structure? ----- When chromatin is released from nuclei and prepared at physiological ionic strength, a fiber of 30 nm thickness is observed. ---- H1 linker histones and the long, flexible tails of core histones have been implicated in forming the higher level of chromatin structure. ---- The 30 nm fiber increases the DNA-packing ratio an additional 6-fold.

Generality of tRNAs: modification, 3' end, two-dimensional and tertiary structure.

-Protein is synthesized through a process that is referred to as translation. Transfer RNAs (tRNAs) function as adaptors to recognize a particular codon in the mRNA and bring a specific AA (as an AA-tRNA) according to the codon, which leading to the synthesis of a polypeptide. -The structure of tRNAs --In 1965, after 7 years of work, Robert Holley (Cornell University) published the base sequence of a yeast tRNA that carries AA alanine. 1. 77 nucleotides; 2. 10 of the 77 nucleotides are modified from the standard 4 nucleotides of RNA (A, G, C, U). -The generality of tRNAs 1. Roughly the same length: between 73 and 93 nucleotides; 2. A significant percentage of the bases are enzymatically modified posttranscriptionally; 3. tRNAs have a two- dimensionalstructure as a cloverleaf. 4. The 3' end of the tRNA molecule is always a adenosine (A). 5. Tertiary structure: two double helices arranged in the shape of an L.

The dynamic nature of rRNAs, tRNAs, hnRNAs, and mRNAs.

-Pulse-chase results 1. Most of the RNA in the cell is present as 18S and 28S rRNA 2. mRNA and hnRNA are the major part of newly synthesized RNA. 3. The half-lives of mRNA range from ~15 min to a period of days. 4. tRNAs and rRNAs have half-lives ranging from days to weeks.

RNA polymerases and their functions?

-RNA polymerases I synthesizes larger (28S, 18S, and 5.8S) ribosomal RNAs. -RNA polymerases II synthesizes messenger RNAs and most small nuclear RNAs (snRNAs and snoRNAs). -RNA polymerases III synthesizes various low-molecular-weight RNAs, including transfer RNAs, the 5S ribosomal RNA, and U6 snRNA. ---The transcription in eukaryotes requires a large variety of accessory proteins (transcription factors). ---RNAs (mRNAs, rRNAs, and tRNAs) are transcribed as a considerably longer precusor RNA (primary transcript or pre-RNA). The corresponding segment of DNA is a transcription unit. The primary transcript is processed into smaller, functional RNAs. ---Plants have two additional RNA polymerases (IV and V) that are not essential for life and synthesize siRNAs.

Splicing of pre-mRNA: conserved sequences for RNA splicing.

-RNA splicing: a process that removes introns from pre-mRNA -Conserved sequences for RNA splicing 1. The 5' splice site: G/GC 2. The 3' splice site: AG/G 3. The polypyrimidine tract 4. The preferred nucleotides in the adjacent regions of the intron 5. Exonic splicing enhancers (ESEs): specific sequences situated in the exons

Difference between prokaryotic and eukaryotic DNA replication.

-Replication in eukaryotic cells ---Eukaryotic cells replicate their genome in small portions called replicons. Each replicon has its own origin from which replication forks proceed outward in both directions. ---Autonomous replicating sequences (ARSs): sequences promote replication of the DNA in which they are contained. -Origin recognition complex (ORC): a multisubunit complex specifically binds to the core element of an ARS.

Chromosome scaffold, codensin and cohesion. Kinetochore, its structure and function.

-Scaffold proteins: a structural framework of nonhistone proteins that maintain the basic shape of the intact chromosome. During interphase, the scaffold proteins are dispersed within the nucleus, forming part of the nuclear matrix. -Condensin: a multisubunit protein complex that binds to DNA and helps DNA to form positively supercoiled loops. -Cohesin: a multisubunit protein complex that associates with DNA along its length and holds the two sister chromatids together in G2. -----Most of the cohesin dissociates from the chromosome arms as they become compacted during prophase. -----The cohesin at the centrosomes is released at anaphase. -Centromere is the primary constriction of a mitotic chromosome. -Kinetochore is a proteinaceous, button-like structure at the outer surface of the centromere of each chromatid 1. The site of attachment of the chromosome to the dynamic microtubules of the mitotic spindle; 2. The residence of several motor proteins involved in chromosome motility; 3. A key component in the signaling pathway of an important checkpoint.

What is nucleolus? What genes are inside the nucleoli? The components of nucleoli.

-The DNA sequences encoding rRNA are called rDNA, which are normally repeated hundreds of times and are clustered in one or a few regions of genome. The clusters of rDNA are gathered together as part of one or more irregular shaped nuclear structures (nucleolus/nucleoli). -The nucleoli function in producing ribosomes. The bulk of a nucleolus is composed of ribosomal subunits that give the nucleolus a granular appearance. ----fc contains rDNA ----dfc contains the nascent pre-rRNA transcripts and associated proteins ----gc contains ribosomal subunits in various stages of assembly

Replication of chromatin structure (histones).

-The assembly of DNA onto nucleosomes is a very rapid event. The nucleosomes on both daughter duplexes are very near the replication fork. -The (H3H4)2 tetramers present prior to replication remain intact and are distributed randomly between the two daughter duplexes. The two H2A/H2B dimers of a nucleosome separate from one another and bind randomly to the new and old (H3H4)2 tetramers present on the daughter duplexes.

Helicase, single-stranded DNA-binding proteins, and DNA polymerases.

-The machinery operating at the replication fork: --Helicase (DNA unwinding enzyme): uses energy from ATP hydrolysis to unwind a DNA duplex and exposes the single-stranded DNA templates. --Single-stranded DNA-binding (SSB) proteins: selectively bind to single-stranded DNA, keeping it in an extended state and preventing it from becoming rewound.

Components/factors required for protein synthesis (translation). Three distinct activities (stages).

-The most complicated synthetic activity in a cell, involving: various "charged" tRNAs ribosomes a mRNA numerous proteins with different functions cations GTP -The synthesis of polypeptide chain includes three distinct activities: Initiation-Eukaryotic cells require at least 12 initiation factors comprising a total of more than 25 polypeptide chains. Elongation- For each cycle of elongation, at least two molecules of GTP are hydrolyzed: one during aminoacy-tRNA selection and one during translocation. termination-When a ribosome reaches one of the stop codons (UAA, UAG, or UGA), the signal is read to stop further elongation and release the polypeptide

1. The structure of nucleus: the nuclear envelope, nuclear pore complex and other contents inside the nucleus: nuclear lamina, nucleoplasm, nucleolus, chromatin, and nuclear matrix.

-The nucleus of a eukaryotic cell 1. Nuclear envelope- a boundary between the nucleus and cytoplasm. 2. Chromosomes: highly extended nucleoprotein fibers (chromatin). 3. One or more nucleoli: irregular shaped electron-dense structures that function in the synthesis of rRNA and the assembly of ribosomes. 4. nucleoplasm: fluid substance in which the solutes of nucleus are dissolved. 5. Nuclear matrix: a protein-containing fibrillar network. -The nuclear envelope 1. Two membranes arranged parallel to one another and separated by 10 to 50 nm. -The outer membrane ---The outer membrane ---The inner membrane -The inner membrane 2. Nuclear pores -The nuclear lamina ----The nuclear lamina is a thin filamentous meshwork, which provides mechanical support to the nuclear envelope and serves as a site of attachment for chromatin fibers at the nuclear periphery. -The nuclear pores are the gateways across the nuclear envelope barrier, which allow passage of macromolecules between the nucleus and cytoplasm. 1. Large numbers of proteins that are synthesized in the cytoplasm are transported across the nuclear envelope to participate the replication and transcription of genetic material in the nucleus. 2. The mRNAs, tRNAs, and ribosomal subunits that are manufactured in the nucleus are transported through the nuclear envelope into the cytoplasm. 3. Some components, such as the snRNAs of the spliceosome, are synthesized in the nucleus, assembled into RNP particles in the cytoplasm, and then shipped back to the nucleus to function in mRNA processing. --Nuclear pores contain a complex, basket-like apparatus called the nuclear pore complex (NPC) that appears to fill the pore like a stopper, projecting into both cytoplasm and nucleoplasm. -The nuclear matrix -- The nuclear matrix is an interconnected network of proteinaceous fibrils throughout the nuclear space. 1. A skeleton to maintain the shape of the nucleus. 2. A scaffold to which the chromatin is attached. 3. To anchor that machinery that is involved in the various activities of the nucleus.

Plant cell plate and its formation.

-The phragmoplast consists of clusters of interdigitating microtubules orientated perpendicular to the future plate. 1. The vesicles fuse with each other to form an interwoven tubular network. 2. New vesicles continue the process of tubule formation and fusion, which extends the network on an outward direction. 3. The leading edge of the growing network contacts the parent PM at the boundary of the cell. 4. The tubular network matures into a continuous, flattened partition. The membranes of the network becomes the PMs of the two daughter cells, whereas the secretory products form the cell plate.

Spindle checkpoint.

-The spindle checkpoint: operates at the transition between metaphase and anaphase. -If a chromosome fails to become aligned properly at the metaphase, the checkpoint mechanism delays the onset of anaphase. -The kinetochores of mislocated chromosomes contain a protein complex Mad2, which binds to the APC activator Cdc20. The APC complexes are unable to ubiquitinate securin that keeps all of the sister chromatins attached to one another by conhesin.

Aminoacyl-tRNA synthetase (aaRS).

-There are 20 different aminoacyl- tRNA synthetases. -Each of the synthetases is capable of "charging" all of the tRNAs that are appropriate for one AA. -Aminoacyl-tRNA is synthesized by a two-step reaction: 1. ATP + AA aminoacyl-AMP + PPi 2 Aminoacyl-AMP + tRNA aminoacyl-tRNA + AMP -The aminoacyl-tRNA synthetases determine which AA is linked to a particular tRNA.

Compare prokaryotic DNA polymerases with eukaryotic DNA polymerases. What is the function of each DNA polymerase?

-Three DNA polymerases in bacteria: 1. DNA polymerase III: polymerizing activity and 3'5' exonuclease activity -part of a large protein complex called DNA polymerase III holoenzyme (replisome), synthesizing DNA strands during DNA replication. -DNA polymerase III holoenzyme (replisome): a multisubunit complex 2. DNA polymerase II: polymerizing activity and 3'5' exonuclease activity -Unknown functions 3. DNA polymerase I: polymerizing activity, 5'3' exonuclease activity, and 3'5' exonuclease activity -a single polypeptide, having three activities in different domains of the polypeptide, functioning in DNA repair and removing the RNA primers of Okazaki fragments and replacing them with DNA. -Eukaryotic DNA polymerases ----Five classic DNA polymerases: alpha, beta, gamma, delta, epsilon 1. Polymerase alpha is tightly associated with primase, and together they initiate the synthesis of each Okazaki fragment. 2. Polymerase beta functions in DNA repair. 3. Polymerase gamma (y) replicates mitochondrial DNA. 4. Polymerase delta is the primary DNA-synthesizing enzyme during replication. 5. Polymerase epsilon (e) is unclear, maybe involved in nuclear DNA replication. -Other DNA polymerases: eta (n), Kapa (k) and iota (i) have a specialized function that allows cells to replicate damaged DNA. -All of the eukaryotic polymerases elongate DNA strands in the 5'3' direction. Polymerases gamma, delta, and epsilon have 3'5' exonulease activity.

What is cell cycle?

-Three broad categories of cells 1. Cells that are highly specialized and lack ability to divide 2. Cells that normally do not divide but induced to divide 3. Cells that normally have relatively high dividing activity -Cell cycle: the cycle of cell growth, replication of the genetic material and nuclear and cytoplasmic division. --Interphase: G1 (first gap), S phase, G2 (first gap) --M phase: Mitosis, cytokinesis

Anaphase A and B.

-Two separate but simutaneous movements: 1. Anaphase A The movement of the chromosomes toward the poles. The net loss of subunits at the minus ends and losses of subunits at the plus ends of the chromosomal microtubules result in shortening of the chromosomal fibers. 2. Anaphase B The two spindle poles move further apart. The net addition of subunits to the plus ends of the polar microtubules elongates the mitotic spindle.

What is unwinding problem? How is it solved? DNA gyrase.

-Unwinding strands cause torsional stress: unseparated portion becomes more tightly wound. - DNA ahead of the replication machinery becomes overwound and accumulates positive supercoils - DNA gyrase: a type II topoisomerase -- DNA gyrase relieves the mechanical strain that builds up during replication: 1. cleaving both strands of the DAN duplex; 2. passing a segment of DNA through the double-stranded break to the other side; 3. sealing the cuts. -The process is driven by the energy released by ATP hydrolysis.

How did cell fusion experiments demonstrate the presence of key factors that regulate the transition from G1 to S and G2 to M? What is premature chromosomal compaction?

-When G1-phase and S-phase cells were fused, the G1-phase nuclei initiated DNA synthesis; -When G2-phase and S-phase cells were fused, the G2-phase nuclei did not initiate another round of DNA synthesis. -Conclusions: 1. the cytoplasm of a replicating cell contains diffusible factors that stimulate initiation of DNA synthesis in G1-phase nuclei; 2. the G2-phase nuclei can no longer respond to initiation factors present in the S-phase cell cytoplasm. ----suggesting that the transition from G1 to S was induced by cytoplasmic factors. -When G1-phase and M-phase cells were fused, the G1-phase chromatin underwent premature chromosomal compaction (PCC) to form a set of elongated compacted chromosomes. -When G2-phase and M-phase cells were fused, the G2-phase chromosomes underwent PCC to form doubled compacted chromosomes. -When S-phase and M-phase cells were fused, the S-phase chromosomes underwent PCC to form pulverized chromosome fragments. -Conclusions: The cytoplasm of an M-phase cell contains diffusible factors that stimulate premature chromosomal compaction of G1-phase, G2-phase, and S-phase chromosomes, suggesting that the transition from G2 to M was induced by the cytoplasmic factors.

Nucleocytoplasmic exchange: nuclear localization signal (NLS) and nuclear export signal (NES).

-While low-molecular-weight solutes penetrate the nuclear pores by simple diffusion, macromolecules, such as proteins and RNAs, pass through the central channel of the NPC with the aid of special transport systems -Ions, metabolites, and proteins <20-40kD in size can passively diffuse through 90-A aqueous channels that span the NPC. -Nuclear localization signal (NLS) ----In 1982, Robert Laskey and his coworkers (Medical Research Council of England) found that nucleoplasmin (one of the abundant nuclear proteins of amphibian oocytes) contains a stretch of AAs near its C-terminus that functions as a nuclear localization signal (NLS). ----NLSs consist of one or two short stretches of positively charged AAs, such as the T-antigen encoded by the virus SV40 contains an NLS identified as -Pro-Lys-Lys-Lys-Arg-Lys-Val-. -Transport receptors: Importins Exportins -Ran-GTP functions in both import and export of macromolecules ---Whereas Ran-GTP induces the disassembly of imported complexes, it promotes the assembly of exported complexes. -Proteins exported from the nucleolus contain AA sequences (nuclear exporting signals, or NESs) that are recognized by transport receptors that carry them through the nuclear envelope to the cytoplasm.

Holliday junction and how is it resolved?

1. Align two homologous DNA molecules 2. Nick the DNA at the same place on the two molecules (with same polarity) 3. Exchange strands and ligate --- Holliday junction 4. Branch migration ------ Resolve the structure - Noncrossover - Heteroduplex - Crossover

Why is nucleus an organized organelle?

1. Chromosomes are concentrated into a distinct territory that does not overlap extensively with the territories of other chromosomes. 2. The portions of chromosomes that are not attached to the nuclear envelope are capable of moving randomly within a restricted zone of the nucleoplasm. 3. The RNA processing machinery is not spread uniformly throughout the nucleus, instead, it is concentrated within irregular "speckles" domains.

What are the properties of genetic code?

1. How many nucleotides define one codon for one AA? -Nucleotides: A, T, G, C -Amino acids: 20 -4^1=4, 4^2=16,4^3=63... 2. Are the genetic codons overlapping with each other? -Genetic code: Gorge Gamow's proposal 1. Each AA in a polypeptide was encoded by three sequential nucleotides or the codons for AAs were nucleotide triplets. 2. The code was overlapping (this was a hypothesis). -Identifying the codons -A technique developed by Marshall Nirenberg and Heinrich Matthaei -Poly(U): 5'-UUUUUUUUUUUU---3' 20 AAs The materials necessary for protein synthesis- Polyphenylalanine ----Therefore, UUU specifies phenylalanine 1. Codons specify the same AA are clustered within a particular portion of the chart. 2. 2. Codon assignment are such that similar AAs tend to be specified by similar codons. 3. The greatest similarities between AA-related codons occur in the first two nucleotides of the triplet, whereas the greatest variability occurs in the third nucleotide. The genetic code is degenerate.

Transcription and processing of 5S rRNA (about 120 nt)

1. In eukaryotes, the 5S rRNA molecules are encoded by a large number of identical genes: -separated from other rRNA genes; -located outside the nucleolus; -organized in tandem array; -alternating with nontranscribed spacers; -the promoter sequence lies within the coding section of the gene. 2. The 5S RNA genes are transcribed by RNA polymerase III. RNA polymerase III can bind to a promoter site located within the transcribed portion of the target gene. 3. The 5' end of the primary transcript is identical with that of the mature 5S RNA, but the 3' end usually contains extra nucleotides that removed during processing. 4. After synthesis, the 5S RNA is transported to the nucleolus to join the other components involved in the assembly of ribosomal subunits.

Properties of heterogeneous nuclear RNAs (hnRNAs).

1. Large molecular weights (up to 80S, 50,000 nt); 2. RNAs of diverse (heterogeneous) nucleotide sequence; 3. Only in the nucleus.

Synthesis and processing of mRNA.

1. Promoter 2. General transcription factors (GTFs) 3. RNA polymerase II: a complex composed of 12 subunits 4. A TATA box (very similar to 5'-TATAAA-3'): between -24 and -32 1. All eukaryotic mRNA precursors are synthesized by RNA polymerase II, a complex composed of 12 different conserved subunits. 2. Besides RNA polymerase II, a number of general transcription factors (GTFs) required to initiate transcription. 3. Promoters for RNA polymerase II lies to the 5' end of each transcription unit. 4. A TATA box (very similar to 5'-TATAAA-3') lies between 24 and 32 bases from the transcription start site. The TATA box is the of assembly of a preinitiation complex that contains the GTFs and RNA polymerase II.

How is the mitotic spindle formed? What is chromosome congression.

1. Replication of centrosome: initiated at the G1-S transition. A. The two centrioles move apart within the centrosome; B. A daughter centriole is assembled next to each maternal centriole; C. The centrosome splits into two adjacent centrosomes. 2. Formation of astral microtubules around each centrosome during early prophase. 3. The two centrosomes move toward opposite ends of the cell through elongation of polar microtubules, establishing the two poles of a bipolar mitotic spindle.

What is a split gene? Introns and exons.

1. Split genes: genes with intervening sequences. 2. Exons: those sequences of a split that are present in the mature RNA. 3. Introns: the intervening sequences. Introns are found in all types of eukaryotic genes, including tRNA, rRNA, and mRNA genes.

Components of ribosomes.

1. The interface between the small and and large subunits forms a relatively spacious cavity that is lined almost exclusively by RNA. 2. The active site, where AAs are covalently linked to one another, also consists of RNA. 3. The mRNA is situated in a narrow channel that winds around the neck of the small subunit, passing through the A, P, E sites. 4. A tunnel runs completely through the core of the large subunit beginning at the active site.

Structure of ribosome: A (aminoacyl), P (peptidyl), and E (exit) sites. Where and how peptide is formed at these three sites?

1. The interface between the small and and large subunits forms a relatively spacious cavity that is lined almost exclusively by RNA. 2. The active site, where AAs are covalently linked to one another, also consists of RNA. 3. The mRNA is situated in a narrow channel that winds around the neck of the small subunit, passing through the A, P, E sites. 4. A tunnel runs completely through the core of the large subunit beginning at the active site.

Mechanism restricting replication once per cell cycle.

1. The origin of replication is bound by an ORC complex; 2. "Licensing factors" bind to the ORC to assemble a prereplication complex. 3. Activation of key protein kinases leads to the initiation of replication. The activity of Cdk, a cyclin dependent kinase, remains high from S phase through mitosis, which suppresses the formation of new prereplication complex. 4. The "licensing factors" move with the replication fork to help complete replication of a replicon.

Transcription and processing of tRNAs.

1. There are about 50 different species of tRNA, each encoded by a repeated DNA sequence: -tRNA genes are found in small clusters scattered around the genome; -a single cluster contains multiple copies of different tRNA genes; -the DNA sequence encoding a given tRNA is found in more than one cluster; -in a cluster, tRNA genes are separated by nontranscribed spacers; -the promoter sequence lies within the coding section of the gene. 2. tRNAs are transcribed by RNA polymerase III. 3. The primary transcript of a tRNA is larger than the final product, and pieces on both the 5' and 3' sides of the pre-tRNA (and a small interior piece in some cases) are trimmed away. 4. All mature tRNAs have the triplet sequence CCA at their 3' end, which are encoded in the tRNA gene in prokaryotes and added enzymatically after the tRNA is processed in eukaryotes.

If the DNA content of a mother cell is 24C, after meiosis I, the DNA content of each daughter cell will be ________ .

24C

Bacteria are grown in a medium containing 15NH4Cl for a number of generations so that all of their genomes are made of fully "heavy" DNA. The bacteria are then moved to a new medium and grown in 14NH4Cl. After one generation time, what does the DNA look like? 1. All of the DNA is made of 2 "light" strands. 2. All of the DNA is made of 2 "heavy" strands. 3. All of the DNA is made of 1 "heavy" strand and 1 "light" strand. 4. Each strand is made of a mixture of "heavy" and "light" DNA, with each strand being between 75% and 100% "light". 5. Half of the DNA is made of 2 "light" strands and half of the DNA is made of 2 "heavy" strands.

3

Which of the following DNA molecules could serve as an effective template for DNA synthesis? 1) an intact, linear, double-stranded DNA 2) a single-stranded, circular DNA 3) a partially double-stranded DNA 4) an intact, linear single-stranded DNA

3

What happens if histone H1 is selectively extracted from compacted chromatin (30 nm fibers)?

30-nm fibers uncoil to form a thinner, more extended beaded filament.

Based on the Wobble Hypothesis, the anticodon 5'IAU3' will not recognize which of the following codons?

5'AUG3'

All DNA polymerases lay down nucleotides in a ______ direction and move along the template in a _______ direction.

5'—>3', 3'—>5'

What is responsible for joining eukaryotic Okazaki fragments together?

DNA ligase

How does genetic information flow? Transcription and translation.

Flow of information in eukaryotic cells -Transcription: synthesis of an RNA from a DNA template. -Translation: synthesis of a protein from a mRNA molecule.

Which of the following binds to the TATA box?

TBP

If you fuse a G1 cell to a cell in the M phase, which of the following responses will occur ?

The G1 nucleus will undergo premature chromosomal condensation to form a set of compacted chromosomes.

Anticodon: direction, wobble hypothesis.

The anticodon: a stretch of three sequential nucleotides in the tRNA that participates in the specific interaction with the codon of the mRNA. The anticodon is located in the middle loop of the tRNA molecule. This loop is invariably composed of 7 nucleotides, the middle 3 of which constitute the anticodon. -Wobble hypothesis: proposed by Francis Crick -The base pairing rules governing the wobble at the third position of the codon: -----Anticodon: U, G, I (inosine) -----Codon: A, G; U, C; U, C, A

Which of the following is NOT a normal property of eukaryotic mRNAs?

They are found in the cytoplasm and inside the Golgi complex

Transcription and processing of 28S, 18S and 5.8S rRNAs.

Three rRNAs (the 28S, 18S, and 5.8S) are synthesized from a single primary transcript. The 5S rRNA is synthesized from a separated pre-RNA outside the nucleolus.

Once the interconnected Holliday junctions are formed, the structure must be resolved and restored to two discrete DNA molecules through another round of DNA cleavage. Which of the following statements describes a possible product of the resolution? a. the two DNA molecules contain only short stretches of genetic exchange. b. Part of one DNA molecule is covalently joined to part of the homologous DNA molecule, creating a site of genetic recombination. c. No effective crossover is created. d. A crossover is created.

a+b+c+d

How does meiosis raise the genetic variability in a population of organisms from one generation to the next? a. Independent assortment allows maternal and paternal chromosomes to be shuffled as gametes form. b. Genetic mutations caused by deletions. c. It changes the number of chromosomes in the organisms from generation to generation. d. Genetic recombination allows maternal and paternal alleles on a given chromosome to be shuffled.

a+d

In meiosis, during which process do the paired homologous chromosomes separate?

anaphase I

Spliceosomes are macrocolecular complexes that consist of a variety of proteins and a host of snRNAs. The snRNAs in the spliceosomes ________.

catalytically splice pre-mRNAs

During metaphase I of meiosis, what appears to hold homologues together as a bivalent?

chiasmata

The assembly of the beta-clamp around the DNA requires a multisubunit _______ that is also part of the ________; part of this structure opens the ______ so that it can fit around the DNA and then function to recruit the core polymerase.

clamp loader, DNA polymerase III holoenzyme, β clamp

The DNA strand growing toward the replication fork grows ______ in a 5'—>3' direction as the replication fork advances and is called the ________.

continuously, leading strand

The greatest variability among codons that specify the same amino acid occurs _________.

in the third nucleotide of the triplet

Cell cycle MPF (maturation-promoting factor) contains a subunit with ________ and a ________ subunit.

kinase activity, cyclin

What is the name of the proteins that make up the nuclear lamina and of what structural superfamily are they a member?

lamins, intermediate filaments

Movement past the transition point near the end of G2 that moves cells on to mitosis requires cdc2 activation by _______.

mitotic cyclins

In meiosis, genetic recombination is completed by the end of ________.

pachytene

Nuclear localization sequence (NLS) enables a protein to pass through the nuclear pores and enter the nucleus. The classical NLSs consist of one or two short stretches of ________.

positively charged amino acids

What provides the energy that drives the polymerization of RNA from a DNA template?

ribonucleoside triphosphates (NTPs)

From what materials are the new cell walls of two daughter plant cells derived?

secretory products within the Golgi-derived vesicles that form the cell plate

The macromolecular complex that associates with each intron and splices it is called a(n) _____.

spliceosome

"The end-replication problem" is solved by ________.

telomerase

The separation of the poles of the cell during anaphase is accomplished by _______.

the sliding of overlapping microtubules from opposite poles over one another in opposite directions