Chem 471 (Test 3)

Transcription Produces RNA Molecules from DNA

Almost all RNA molecules are derived from genetic information stored in DNA through transcription.

Chargaff's Rules on the base composition of hydrolyzed DNA:

1) Varies from one species to another. 2) Stay the same between tissues of the same species. 3) Not affected by age, nutrient, and environment. 4) The sum of purines (A+G) equals to the sum of pyrimidines (C+T)

Practice Question: Why is the genetic code said to be "degenerate"? A. The first base in a codon is the most important for coding, followed by the second base, and the third base is least important. B. Each amino acid can be specified by more than one codon. C. Fewer amino acids are coded by the four bases than was expected. D. The less complexity in an organism, the fewer codons are specified. E. The genetic code deteriorates with each replication (generation), unless proofreading functions correct for errors.

B Explanation: An amino acid may be specified by more than one codon, so the code is described as degenerate.

Which base pairs have more H bonds? GC or AT

GC Has higher annealing temperature because of 3 H bonds

Naming convention: Naming Genes and Proteins in Bacteria

Gene = italicized EX: dnaA Protein = non-italicized, first letter capitalized EX: DnaA

Eukaryotic Chromosomes

Genetic material of eukaryotic cells is distributed into chromosomes - the diploid (2n) number depends on the species - each chromosome contains a single, very large, duplex DNA molecule that carries a characteristic set of genes • Human somatic cells have 46 chromosomes

What is the difference between a nucleoside and a nucleotide? (Pyrimidines)

Nucleoside= Base+ sugar Nucleotide= Base+ Sugar+ Phosphate N-β-glycosyl bond: *Attached to the 1' position of ribose or deoxyribose through the N-1 position.

What is the difference between a nucleoside and a nucleotide? (Purines)

Nucleoside= Base+ sugar Nucleotide= Base+ Sugar+ Phosphate N-β-glycosyl bond: *Attached to the 1' position of ribose or deoxyribose through the N-9 position.

Ribose- The Pentose Sugar (know structure and numbering)

OH: Fischer= Right Haworth= Down Fischer= Left Haworth= Up • β anomer: the anomeric -OH & the terminal CH2OH are on the same side.

Where are genes located?

On chromosomes

True or False (PAGE SEQUENCING)? The smaller the fragment, the faster it migrates from the - to the end (+) and it is closer to the 5' end.

True

True or False? All nucleotide bases absorb UV light with a strong absorption near 260 nm.

True

True or False? Phosphodiester bonds are acidic and completely ionized at pH 7, negatively charged.

True

True or False? Three-dimensional structure in RNAs have unusual base pairing patterns.

True

ρ-Independent Termination in E. coli

Two distinguishing features for ρ-independent terminators: 1. Have a self-complementary region that forms a hairpin structure before projected end. 2. A conserved string of three A residues (AAA) that are transcribed into U residues near the 3′ end of the hairpin

Topoisomerases I and II

Type I: break one of the two DNA strands and change the linking number in increments of 1 Type II: break both of the two DNA strands and change the linking number in increments of 2.

Termination Codons

UAA, UAG, and UGA

What is positive and negative supercoiling?

Under-winding = (-) supercoiling Overwinding = (+) supercoiling Relaxed state = no net bending of the DNA axis upon itself

Open-Reading Frame

a reading frame without a termination codon among 50 or more codons.

Crick's adaptor hypothesis

a small nucleic acid could act as an adaptor, binding to both a specific amino acid and the mRNA sequence that encodes the amino acid

Anticodon

a three-base sequence on the tRNA that base pairs with mRNA codon

Codon

a triplet of nucleotides that codes for a specific amino acid (read in a successive, non-overlapping fashion during translation)

Sister chromatids

condensed

How do the strands of DNA run?

anti-parallel (opposite directions)

Amino acid arm

carries a specific amino acid esterified by its carboxyl group to the 2′-OH or 3′-OH group of the A residue at the 3′ end of the tRNA

Anticodon arm

contains the 3-letter anticodon

Protein Homeostasis

controlled protein synthesis and degradation. It is a dynamic process.

Exons

coding DNA segments that contain the genetic information for the amino acid sequence of the polypeptide product.

Tertiary structure

complex folding of large chromosomes or the elaborate folding of tRNA or rRNA structures

Topisomerases

enzymes that under-wind or relax DNA (change the linking number) and are especially important for processes such as replication and DNA packaging.

Chromatin

eukaryotic chromosomal material composed of DNA, RNA, and proteins. Amorphous in G0 and interphase (G1, S, and G2) phases

Consensus sequences are..

formed by certain nucleotides that are particularly common at a given position (shown are sequences of the non-template strand)

Size and Packing: How do we pack all these DNA molecules into our body?

multiple layers and multiple modes of tertiary structure

Missense mutations

mutations in which a single new base pair replaces another - Most common type of mutation - In the 3rd (wobble position), single base substitutions change the encoded amino acid only ~25% of the time

Silent mutations

mutations in which the nucleotide is different but the amino acid is the same (e.g., UUU to UUC, both encode Phe).

What does DNA polymerase I do?

remove the RNA primers (5' to 3' exonuclease activity), and synthesize the DNA segment (polymerase)

How are phosphodiester bonds in nucleotide polymers orientated?

same orientation in both DNA and RNA, 5' end to 3' end

What does DNA Ligase do?

seal the remaining nick of two fragments

Regulatory Sequences

segments of sequences of DNA that have a purely regulatory function

In E.coli, what does Topoisomerase IV do?

separates the catenated circles

Oligonucletide

short (50 or fewer nucleotides) nucleic acid

What are Okazaki fragments?

short DNA fragments that are synthesized during replication of the 'lagging' DNA strand. - linked together by DNA ligase - 150 to 200 nucleotides long in eukaryotes -1,000 to 2,000 nucleotides long in bacteria

Centromere

specific attach points for the mitotic spindle *During cell division: Centromere-Kinetochore (disc-shaped protein)-Mitotic spindle

Nucleosomes

spherical complex of histone proteins with about two turns of DNA wrapped around them

Where does DNA contain genetic information?

stored in its nucleotide sequence

What are deoxyribonucleotides?

structural units of DNA

What are ribonucleotides?

structural units of RNA

DNA Replicase System: Primase

synthesizes RNA primers for DNA polymerization. - 5' --> 3' DNA Synthesis (both strands) - Connect the lagging strand segments

Aminoacyl-tRNA

tRNA attached to an amino acid

Chromosomes

tertiary packaging of DNA

Processivity

the average number of nucleotides added before a polymerase dissociates. DNA polymerases vary greatly in processivity

Separation of Catenanes by Topoisomerase IV: What are catenanes?

two topologically interlinked circular chromosomes (two circular dsDNA molecules tangled)

RNA Polymerase Synthesizes RNA

* Both RNA and DNA polymerase are DNA template dependent. DNA-dependent RNA polymerase requires: 1. DNA template 2. four ribonucleoside 5′- triphosphate [(ATP, GTP, UTP, and CTP) = NTPs] 3. Mg2+ (co-factor) RNA POLYMERASE DOES NOT NEED A PRIMER!!

Quantitative PCR

*The more templates, the faster it takes to reach threshold and a smaller number a cycles. Sample 1 reached plateau faster Threshold: - Arbitrarily defined - The more samples, the sooner the threshold is reached CT: cycle number - when the threshold is first surpassed No template control: - no sample DNA

DNA Polymerase Reaction

- DNA polymerase requires Mg2+ ion. - The reaction is a phosphoryl group transfer. - The 3'-OH group at the primer terminus acts as a nucleophile for the α phosphorus of the incoming dNTP to form the phosphodiester bond.

What are some applications about PCR?

- High sensitivity- detect as little as one DNA molecule. - Controls are essential to eliminate false results due to contamination. 1. cloning 2. epidemiological studies 3. genetic diseases and infections 4. forensic medicine

3-D Structure of tRNAs

- L-shaped or boot-shaped - Unique features afford individual recognition by enzymes and codons.

Semidiscontinuous DNA Synthesis

- Leading strand: continuous synthesis; 5′ → 3′ synthesis proceeds in the same direction as the replication fork movement. - Lagging strand: discontinuous synthesis; 5′ → 3′ synthesis proceeds in the opposite direction of the replication fork movement.

Naming convention: Naming Genes and Proteins in Eukaryotic Systems

- No single convention exists for all eukaryotic systems. - Protein naming is complex and variable. Yeast = three italicized uppercase letters followed by an italicized number EX: COX1 (gene) *whenever you se something italicized, it is a gene Protein= Long common name (e.g., cytochrome oxidase) Same as the gene, with one uppercase and two lowercase letters, followed by a number and the letter "p" EX: Rad51p

DNA Topology During Transcription

- Overwound DNA (positive supercoil) ahead of RNA polymerase. - Under-wound DNA (negative supercoil) behind RNA polymerase (relieved through topoisomerase)

Transcription

- RNA polymerase: occupies ~35 bp of DNA during elongation. - During elongation, the growing RNA strand temporarily base-pairs with the DNA template to form a short hybrid RNA-DNA double helix

Specificity in Transcription: Promoters

- RNA synthesis begins at promoters (= specific sequences in DNA that RNA polymerase binds to ---> VARY between genes), and then starts transcription at specific site.

Rate of Error (DNA replication vs. Transcription)

- Rate of error in transcription: one error for every 104 to 105 ribonucleotides incorporated into RNA. ~ 100,000 x higher than the error rate for replication • Rate of error in DNA replication: one error for every 109 to 1010 nucleotides added.

During Transcription...

- The 5 core subunits remain the same. - The regulatory subunit differs: ~ σ subunit is the regulatory subunit at initiation ~ NusA protein is the regulatory subunit for elongation

Bacterial Chromosome

- The E. coli chromosome is a single, double-stranded circular DNA molecule of 4,641,652 bp - It encodes ~4,300 genes for proteins and >200 genes for structural or catalytic RNAs

Next generation sequencing- pyrosequencing

- The addition of nucleotides is detected with flashes of light. 2. Pulse addition of the dNTPs, one at a time.

How is a primer designed?

- The amplification process is driven by an excess of primers (forward and reverse primers). - Primers bind the templates in a sequence-specific manner (A pairs with T, C pairs with G, etc.). - Primers: 5' to 3'. - Annealing temperature (depends on primer): optimized for specific binding between the primers and the templates.

Transcription vs Replication

- Transcription: DNA-Dependent synthesis of RNA. - Replication: DNA-Dependent synthesis of DNA.

Reversible terminator sequencing (Illumina)

- Uses four different modified deoxynucleotides (A, T, G, and C), each with a particular fluorescent label and a 3′ blocking group. - Detect the addition of "color-coded" nucleotides (fluorescent label) with laser.

What are ribosomes and what are they made of?

- are not organelles, even though they are part of the rough endoplasmic reticulum in eukaryotes. - Found in both prokaryotes and eukaryotes, ribosomes are made of RNAs and proteins.

Three letter names in lowercase reflect what? Capital letters added to abbreviation reflects what?

- function - order of discovery, not enzymatic order

Explain how the Sanger method is based on DNA replication

-Double helix model= antiparallel and complementary (A=T and C=G) -In DNA replication: (start with double and separate into single strand) synthesize a new complementary strand for each of the original strands to obtain two identical copies of the original molecule.

Automated DNA sequencing

-Sanger method -template and primer -Dye-terminator: ddNTPs (each with a different colored fluorescent label) & dNTPS -DNA polymerase catalyzed chain extension and termination

What are the steps in Maxam-Gilbert chemical degredation?

-Start with 4 sets of chemical modifications: 1. G+A Specific 2. G specific 3. C+T Specific 4. C Specific - Each DNA strand is cleaved only once at modified sites. -Reaction products are separated by electrophoresis on a DNA sequencing gel (PAGE). - With 5' end-labeled DNA, the sequence (5' to 3') is read from the bottom to the top.

Sanger sequencing

-ssDNA template and primer 4 sets of reactions: 1.ddATP & dNTPS 2.ddCTP & dNTPS 3.ddGTP & dNTPS 4.ddTTP & dNTPS -Electrophoresis -Detect the DNA fragments by autoradiography -Read from bottom to top of the gel.

DNA Polymerase at Work (1)

1. A template DNA strand guides polymerization. 2. A primer = a strand segment complementary to the template, with a free 3′-OH group to which a nucleotide can be added. 3. Template and primer pair up and bind to the enzyme. 4. Incoming nucleotide binds to the insertion site, selected in part by base-pairing and forms phosphodiester bond.

Protein Synthesis: What are the 5 stages?

1. Activation of amino acids (ATP Mg2+) 2. Initiation (50S ribosomal subunit Initiation factors (IF1, IF2, IF3) GTP Mg2+) 3. Elongation 4. Termination and ribosome recycling 5. Folding and post-translational processing

What are the 2 key prerequisites for life?

1. Biological information 2. Analysis

What are some secondary structures of RNAs?

1. Bulge 2. Internal loop 3. Hairpin

Why is Eukaryotic RNA Polymerase II important?

1. Conserved structure, function, and mechanism between bacterial and eukaryotic RNA polymerases. 2. More complex than its bacterial counterpart. 3. Require additional proteins (transcription factors). 4. RNA polymerase II (Pol II) is central to eukaryotic gene expression, responsible for the synthesis of mRNAs and many ncRNAs. Can recognize 1000s of promoters v Four phases of Pol II catalyzed transcription: assembly, initiation, elongation, and termination.

Characteristics of Protein Synthesis

1. Consume up to 90% of the chemical energy 2. Protein metabolism is regulated at many levels. 3. Tightly controlled process

What are the mechanisms too ensure DNA replication fidelity?

1. Correct pairing and base-pair geometry at the active site 2. Intrinsic 3' to 5' exonuclease 3. A separate enzyme system to repair the remaining mismatched base pairs after replication.

What are the two kinds of pentoses?

1. D-Ribose, an aldopentose (RNA: β-D-ribofuranose.) 2. 2-Deoxy-D-Ribose, and aldopentose (DNA: , β-2′-deoxy-D-ribofuranose

What are the multiple enzymatic activities that DNA polymerases have?

1. DNA polymerization (DNA polymerase) 2. nucleic acid degradation, from the 3' end (3' to 5' exonuclease).

What are the basic principles of DNA replication?

1. DNA replication is semiconservative (each strand provides the template for a new strand) 2. Replication begins at an origin and usually proceeds bidirectionally. 3. DNA synthesis proceeds in a 5′→3′ direction and is semidiscontinuous.

What are the 3 major types of DNA metabolism?

1. DNA replication: copies DNA molecules and passes the "blueprint" of the cell from one generation to the next. 2. DNA repair: maintains the integrity of genetic information. Repair systems revert DNA damages caused by a wide range of insults. 3. DNA recombination: : tightly controlled rearrangement of genetic information to maintain genomic integrity

What are the three key steps for each PCR cycle?

1. Denature dsDNA template by heat 2. Anneal primers to the ssDNA 3. Chain extension (elongation) catalyzed by DNA polymerase (5'è3')

Identify RNA Polymerase Binding Site

1. Footprinting = a technique to identify the sequences bound by a particular protein(e.g., RNA polymerase). 2. Radioactively labeled DNA fragments. 3. Chemical or Enzymatic cleavage (once per strand)

DNA Replicase System: Initiation

1. Initiator proteins bind to the origin. 2. Helicase unwinds DNA. 3. Topoisomerase II (DNA gyrase) relieves torsional strain.

Structure of Nucleotides: What are the three characteristic components?

1. Nitrogenous base (pyrimidine or purine) 2. Pentose 3. one or more phosphate

What are some characteristics of RNA?

1. One strand 2. Complex three dimensional (folded more like globular proteins) 3. Three major classes- mRNA, tRNA, rRNA 4. Some have catalytic activities

What are the products of PCR?

1. Predominantly defined by the region that the primers flank 2. Nearly clonal population of DNA molecules generated in vitro

Steps in Transcription Elongation in E.Coli

1. RNA polymerase binds to the promoter, directed by its σ subunit 2. Forms a closed complex (bound DNA remains double-stranded) 3. • Open box, bound DNA is partially unwound around the -10 region (transcription is initiated ---> primer is not needed) 4. Initiation causes a conformational change. 5. The elongation complex moves away from the promoter (promoter clearance). 6. s subunit falls off once elongation starts. 7. NusA protein replaces s subunit *E. coli RNA polymerase holoenzyme: ü 6 subunits (5 core subunits, and 1 regulatory unit).

RNA Polymerase

1. RNA polymerase does not require a primer to initiate RNA synthesis. 2. Chain elongation requires the free 3'- OH group. 3. RNA polymerase adds ribonucleotide units to the 3′-OH end, building RNA in the 5′→3′ direction. 4. RNA polymerase does not have the 3' to 5' exonuclease activity (proofreading) --> higher error rate

What are the adaptations of PCR?

1. Reverse transcriptase- RNA template, reverse transcription into cDNA, then PCR 2. Quantitate PCR- - Binding of fluorescent probes - Amplification and detection

DNA Replication in Eukaryotic Cells

1. Similar to that in bacteria, yet more complex. - Initiation, elongation, and termination 2. Multiple origins of replication. 3. DNA polymerases: o - DNA polymerization: DNA polymerase d - Primer synthesis: DNA polymerase a - DNA repair: DNA polymerase e

What are the exceptions to the general rule?

1. Some genes code for RNA molecules (i.e., tRNA, rRNA). 2. Many proteins are assembled from more than one subunit, each encoded by different genes. 3. Sometimes, more than one protein can be produced from a single gene.

What are the factors that impact DNA annealing?

1. Temperature (too low: unproductive; too high: denaturing) 2. Length of DNA 3. Content of DNA 4. Salt strength

What are the central requirements for DNA polymerization?

1. Template 2. Primer 3. dNTPs

Conformations of Ribose

1. The pentose ring is NOT planar. 2. Four of the five atoms are nearly in a single plan--- "puckered" • Endo: C-2' or C-3' on the same side of C-5' • • Exo: C-2' or C-3' on the opposite side of C-5' •

Nucleotide Bonds

1. The phosphate is esterified to the 5' carbon. 2. The N-β-glycosyl bond covalently joins the 1' carbon of the pentose to the base (N-9 for purines and N-1 for pyrimidines) ---> formed by the removal of the elements of water

What are the steps after the first cycle of PCR?

1. amplifies the DNA template by a factor of 2 per cycle: 2^N 2. The thermostable DNA polymerase

The amino-terminal tails of the histones:

1. are intrinsically disordered 2. are where most of the histone modifications occur 3. play a key role in forming contacts between nucleosomes in the chromatin

Functions of regulatory sequences

1. denote the beginning or the end of genes 2. influence the transcription of genes 3. initiation points for replication or recombination

What are the four main components of a basic PCR reaction?

1. dsDNA template that contains the target segment 2. A pair of synthetic oligonucleotide primers 3. Deoxynucleoside triphosphates (dNTPs) 4. DNA polymerase (heat stable, maximum enzymatic activity at 75°C to 80°C)

What are the functions of nucleotides?

1. energy carriers (ATP) 2. Enzyme cofactors (coenzyme A) 3. signaling molecules (cAMP, cGMP)

What roles do nucleotides play?

1. energy currency (ATP) 2. essential chemical links in cellular responses to hormones and other extracellular stimuli 3. structural components of an array of enzyme cofactors and metabolic intermediates 4. building blocks of nucleic acids - DNA and RNA

What are the major types of RNA Molecule?

1. messenger RNAs (mRNAs) = encode the amino acid sequences of polypeptides 2. transfer RNAs (tRNAs) = read the mRNA and transfer/carries the appropriate amino acid to a growing polypeptide chain during protein synthesis 3. ribosomal RNAs (rRNAs) = constituents of ribosomes, the cellular machines that synthesize proteins 4. noncoding RNAs (ncRNAs) = have a variety of catalytic, structural, and regulatory functions

What are some characteristics of the double helix model?

1. right-handed double helix 2. hydrophilic backbone, and hydrophilic bases inside 3. 10.5 base pairs/turn 4. Antiparallel, complementary strands 5. held together by H-bonding and base stacking interactions 6. Major and minor groove

What are the 2 classes of termination signals encoded in the DNA sequence?

1. ρ-independent 2. ρ-dependent

What is B-form DNA?

1.)Most stable structure under physiological conditions 2.) Standard for studying DNA properties

What are the steps in next generation sequencing?

1.Break genomic DNA into random fragments that are a few hundred base pairs long. 2. Tag the ends of DNA fragments with synthetic oligonucleotides as references markers. 3. Immobilize the individual fragments on a solid surface. 4. Sequence all DNA clusters (millions) at the same time. 5. Data captured and analyzed by computers.

Automated DNA sequencing (2)

1.Electrophoresis to separate the fragments. 2.Laser beam to activate and detect the fluorescent dyes. 3.Computer generated sequencing data.

What are the three stages of transcription in Bacteria?

1.Initiation 2.Elongation 3.Termination

What is the histone core composed of?

8 histone molecules: two copies each of H2A, H2B, H3, and H4

Practice Question: Which mutation is an example of a transition mutation? A. G→A B. G→T C. A→C D. A→T

A Explanation: A transition mutation is when a purine is replaced by another purine or a pyrimidine is replaced by another pyrimidine.

Practice Question: Which statement about bacterial DNA polymerase I is false? A. It is the principle replication enzyme in E. coli. B. It has both 3′→5′ exonuclease activity and 5′→3′ exonuclease activity. C. It is active as a single subunit.

A Explanation: DNA polymerase I is not the primary enzyme of replication (DNA Polymerase III is) ; instead, it performs a host of cleanup functions during replication, recombination, and repair.

Practice Question: Which factor is NOT associated with the process of transcriptional termination? A. Sequence-dependent binding of Term protein in eukaryotes B. Dephosphorylation of the CTD of Pol II in eukaryotes C. The action of ρ helicase in E. coli D. Hairpin formation in the RNA product of E. coli RNA polymerase

A Explanation: In eukaryotes, termination begins when elongation factors dissociate. The Pol II carboxyl-terminal domain (CTD) is dephosphorylated, a process facilitated by termination factors. The transcription machinery is then recycled, ready to initiate another transcript.

Practice Question: Sanger sequencing was performed on DNA from chloroplasts of a new plant species discovered in the rain forest of Puerto Rico. The x-ray film showed bands in the A, C, and T lanes, but the G lane was blank. What is the MOST likely explanation for this unexpected result? A. ddG was not added to the reaction tube. B. Different template DNA was added to the reaction tube. C. The template DNA contained no G. D. The template DNA contained no C.

A Explanation: ddNTPs interrupt DNA synthesis because they bind to the template strand but lack the 3′-hydroxyl group needed to add the next nucleotide. Omitting ddG from the reaction tube is the most likely explanation for a blank G lane.

DNA Replicase System: Termination

A specialized set of proteins act to terminate DNA replication at specific location - terminus region (Ter).

How many hydrogens does A:T and G:C pairs have?

A:T pair: 2 hydrogen bonds G:C pair: 3 hydrogen bonds

In RNA, what are the base pairs usually symbolized as?

A, G, U, and C

The structure of complementary RNA strands is usually what form?

A-form right-handed double helix

In free form, ribonucleotides are commonly abbreviated as?

AMP, GMP, UMP, and CMP

Initiation Codon

AUG

Purines (know structure and numbering)

Adenine (A)= in DNA and RNA Guanine (G)= in DNA and RNA

Supercoiling: What is the advantage of under-winding?

Advantage: easier strand separation = easier access to genetic information Under-winding ---> Structural strain ---> Supercoiling (requires less energy to accommodate the strain)

Sequence Assembly Requires the Computerized what?

Alignment of Overlapping Fragments

Under _____ conditions, RNA is ______ due to the presence of hydroxyl group at ____.

Alkaline; Rapidly hydrolyzed; C2' *DNA is NOT rapidly hydrolyzed

Double strand DNA is ..?

Antiparallel and complementary

What are the three enzymes and what are their functions in next generation sequencing-pyrosequencing?

Apyrase to degrade the dNTPS that did not react. Sulfurylase to convert the pyrophosphate into ATP. Luciferase to detect the ATP and generate light.

Practice Question: What valuable piece of information is obtained by DNA foot printing? A. The location of a promoter region of DNA B. The specific sequence of bases to which a particular protein binds C. The location of the 5′ end of a coding sequence D. All the DNA sequences in a genome to which a known DNA sequence binds E. The location(s) in genomic DNA to which a known sequence of RNA binds

B Explanation: Foot printing, a technique derived from principles used in DNA sequencing, identifies the DNA sequences bound by a particular protein.

Practice Question: Among the following, which shows a correctly named bacterial gene? A. dnaA B. dnaA C. DnaA D. DnaA

B Explanation: For naming bacterial genes, three italicized lowercase letters reflecting a function, followed by capital letters indicating the order of discovery. In this case, the dnaA gene encodes the DnaA protein

Practice Question: Among the following, which correctly describes the reaction catalyzed by DNA polymerase? A. The nucleophile is the α phosphate of the incoming deoxynucleoside 5′-triphosphate. B. The nucleophile is the 3′-OH group of the nucleotide at the 3′ end of the growing strand. C. The nucleophile is the 3′-OH group of the nucleotide at the 5′ end of the growing strand. D. The nucleophile is the γ phosphate of the incoming deoxynucleoside 5′-triphosphate.

B Explanation: The 3′-OH of the nucleotide at the 3′ end of the strand (the nucleophile) attacks the α phosphorus of the incoming dNTP.

Practice Question: Other than having U in the RNA in place of T in the DNA, the sequence of transcribed RNA: A. is identical to the template DNA strand sequence. B. is identical to the non-template DNA strand sequence. C. is the reverse of the template DNA strand sequence. D. is the reverse of the non-template DNA strand sequence

B Explanation: The strand that serves as template for RNA synthesis is called the template strand. The DNA strand complementary to the template, the non-template strand, or coding strand, is identical in base sequence to the RNA transcribed from the gene, with U in the RNA in place of T in the DNA. - Anti-parallel - Complementary

Naming Convention

Base --> Nucleoside (-sine) --> Nucleotide (-nylate) 1. Adenine- adenosine; adenylate (RNA) 2. Guanine- guanosine ; guanylate (RNA) 3. Cytosine- cytidine; cytidylate (RNA) 4. Thymine- thymidine; thymidylate (DNA) 5. Uracil- uridine; uridylate (RNA) NOTE: If the sugar is deoxyribose, then add "deoxy" in front of the nucleoside or nucleotide. For uridine and uridylate, there is no "deoxy" forms.

Base-Pair Geometry in DNA Replication

Base pairing: A=T and G=C Incorrectly paired nucleotide can be rejected before the phosphodiester bond is formed.

What are the breaks caused by in RNA structures?

Breaks caused by mismatched or unmatched bases result in bulges or internal loops.

Practice Question: Which term describes the production of two or more related but distinct proteins from a single transcript? A. transition mutation B. third base wobble C. translational frame-shifting D. RNA editing

C Explanation: A few genes are structured so that ribosomes "hiccup" at a certain point in the translation of their mRNAs, changing the reading frame from that point on. This process, called translational frameshifting, allows two or more related but distinct proteins to be produced from a single transcript.

Practice Question: How many codon combinations actually encode amino acids? A. 20 B. 32 C. 61 D. 64

C Explanation: Among the 64 possible codons, 61 encode amino acids, and the other three are termination codons.

Practice Question: The lagging strand in DNA replication: A. requires its own replisome. B. is synthesized after the leading strand. C. is an inescapable consequence of replicating both strands of template DNA at a single replication fork. D. causes the formation of Okazaki fragments in the leading strand.

C Explanation: In the leading strand, 5′→3′ synthesis proceeds in the same direction that the replication fork moves. In the lagging strand, 5′→3′ synthesis proceeds in the direction opposite to the direction of fork movement.

Practice Question: Nucleosomes consist of: A. chromosomes in a nucleus B. only DNA C. DNA and histones D. organelles found in the nucleus

C Explanation: Nucleosomes consist of DNA wrapped tightly around a core of eight histone molecules.

What are dideoxy NTPs (ddNTPs)?

Chain terminators. When a ddNTP is inserted in place of a dNTP, strand elongation is halted because it lacks the 3′-hydroxy group needed for the next step nucleotide addition.

Pyrimidine (know structure and numbering)

Cytosine= in DNA and RNA Thymine (T)= only in DNA Uracil (U)= only in RNA

Practice Question: What other name can be given to a nucleotide? A. glycosylated nucleoside B. purinated pentose C. deoxyribonucleotide D. nucleoside phosphate

D Explanation: A nucleotide has three characteristic components: (1) a nitrogenous (nitrogen-containing) base, (2) a pentose, and (3) one or more phosphates. The molecule without a phosphate group is called a nucleoside. Thus, a nucleoside phosphate is another name for a nucleotide.

Practice Question: What type of bond must be formed between Okazaki fragments to generate a complete DNA strand? A. hydrogen B. disulfide C. ester D. phosphodiester E. ionic

D Explanation: DNA ligase catalyzes the formation of a phosphodiester bond between a 3′ hydroxyl at the end of one DNA strand and a 5′ phosphate at the end of another strand.

Practice Question: Which method is NOT associated with DNA sequencing? A. Sanger sequencing B. electrophoresis C. alignment D. Maxam and Watson sequencing

D Explanation: Sanger sequencing enables the sequencing of large DNA molecules. The different-sized fragments formed during the Sanger method of DNA sequencing are separated by electrophoresis. With more advanced DNA sequencing technologies, translating the sequences of millions of short DNA fragments into a complex and contiguous genomic sequence requires the computerized alignment of overlapping fragments.

Practice Question: Which statement about general transcription factors is false? A. They are required at every Pol II promoter. B. They are usually designated TFII with an additional identifier. C. They are highly conserved in all eukaryotes. D. They are proteins that block the synthesis of RNA at specific genes.

D Explanation: The general transcription factors required at every Pol II promoter (factors usually designated TFII with an additional identifier) are highly conserved in all eukaryotes. Repressors are proteins that block the synthesis of RNA at specific genes.

Practice Question: Mature tRNAs: A. vary in size from 50 to 100 nucleotides. B. have an anticodon arm, a D arm, an amino acid arm, and a ribosomal arm. C. have little secondary structure. D. All have very similar three-dimensional structures

D Explanation: Transfer RNAs are relatively small (73 and 93 nucleotide residues) and consist of a single strand of RNA folded into a precise three-dimensional structure.

Eukaryotic Transcription: What are the 3 kinds of nuclear RNA polymerases?

DIFFERENT CLASSES RESPONSIBLE FOR DIFFERENT JOBS!

What are the two types of nucleic acids?

DNA and RNA Sugar!: 2'-deoxy-D-ribose. --> DNA D-ribose --> RNA

What enzyme catalyzes chain extensions?

DNA polymerase In the picture: The 3′-hydroxyl group of the primer reacts with an incoming dNTP to form a new 3',5'-phosphodiester bond.

What is the principal replication enzyme in E.coli?

DNA polymerase III i

DNA Polymerase at Work (2)

DNA polymerase active site has two parts: 1. insertion site = where the incoming nucleotide binds 2. Post-insertion site = where the new base pair resides when the polymerase slides forward - Newly formed base pair translocates to the postinsertion site.

What are genes and the general rule?

DNA segments that contain the coding sequences for protein and RNA molecules; one gene equals polypeptide.

Telomeres

DNA sequences at the ends of chromosomes that help to stabilize the chromosomes (eukaryotic chromosomes end in these)u

What is denaturation?

Denaturation/unwinding: disruption of H-bonds and base stacking. Double helix to two single strands.

Reversible Denaturation and Renaturation of DNA

Denaturing conditions: pH: 7.0 to extremes Temp: 25°C to >80°C •

Direction of Transcription

Double-stranded DNA unwind ---> transcription (DNA/RNA hybrid) ---> RNA falls off and the 'used' DNA rewinds to dsDNA = transcription bubble moves along the DNA

What does the free 3' OH serve as in DNA?

Elongation point

True or False: Only half of the chromosome is usually copied during replication.

False: The entire chromosome is usually copied during replication.

True or False? dNTP cannot be dATP, dCTP, dGTP, and dTTP

False; it is a mixture of all 4 types

The Central "Dogma" of Biochemistry

Flow of genetic information: DNA → RNA → Protein

What are the histone 5 major classes from largest to smallest?

H1, H3 (has nearly identical amino acid sequences as H4), H2A, H2B, H4

What is the easiest way to denature DNA?

Heat

The base pairing between codon and anticodon occurs _____ , and is ______.

Hydrogen bonding, anti-parallel

Frame-Shift

If you don't start at the right codon, the reading frame will be wrong, and all the amino acids will be wrong ones.

Topoisomerases as Drug Targets

In bacteria: Inhibitors of bacterial topoisomerases kill bacteria and are used as antibiotics to treat infection. - Type I (Topo I and III): relaxes DNA and increases Lk - Type II (Topo II or DNA gyrase): introduces negative supercoils and decreases Lk

Error Correction by DNA Polymerases (2)

Incorporation of the correct deoxynucleotide

DNA Under-winding and Superhelical Density Equations

Linking number (Lk): topological property of dsDNA. - For closed circular DNA: Lk = length / 10.5 - DNA underwinding: Lk0 for relaxed DNA, ΔLk = Lk - Lk0 - Superhelical density: (s): s = ΔLk /Lk0

Replication fidelity is essential for?

Maintaining genome integrity (one error every 109 to 1010 nucleotides added (equals to one error per 1,000 to 10,000 replications in E. coli).

______ from one generation to another ensures species continuity.

Maintenance and transmission of genetic information

Nucleotides are _______ and nucleic acids are ________.

Monomers; polymers

Proofreading by DNA Polymerase

Nucleases = degrade both DNA and RNA. DNases = enzymes that degrade DNA. Exonucleases = degrade nucleic acids from the end (5' to 3' or 3' to 5'). Endonucleases = begin to degrade at specific internal sites. ALL DNA POLYMERASES HAVE 3' TO 5' EXONUCLEASE ACTIVITY.

Are nucleotides soluble in pure water?

No. Nucleotides are hydrophobic and relatively insoluble in pure water (pH 7.0). *They are more charged and soluble at acidic or alkaline pH values.

Defining DNA Strands at the Replication Fork

New DNA strands are always synthesized in the 5′→3′ direction. The template is always read in the 3′→5′ direction.

dsDNA

One strand of the dsDNA contains the coding information that is used to make protein (coding strand) The other strand of the dsDNA is used as a template to make the mRNA (template strand) Each amino acid of a polypeptide chain is coded for by three consecutive nucleotides in a single strand of DNA (codon)

What are the hydrophilic backbones composed of in phosphodiester bonds?

Phosphate and pentose residues

DNA Templates in Transcription

RNA: 1. One DNA template strand 2. 5' to 3' synthesis 3. 'U' in RNA, in place of 'T' in the DNA coding strand sequence

Nucleotides

Play a variety of roles in cellular metabolism.

______ genes don't have introns, but _______ genes contain both exons and introns.

Prokaryotic; Eukaryotic

RNA Metabolism: What is RNA? What is its function?

RNA (as a collective group) is considered the only macromolecule known to function in both: 1. Storage and transmission (= carrier) of genetic information: - Classical central dogma: DNA -->RNA --> Protein 2. Catalysis: Catalytic RNA or ribozyme -Ribozymes = catalytic RNAs that act as enzymes

Phosphorylation of the CTD of RNA Polymerase II

RNA polymerase II has a long c terminal domain and it oxidizes during transcription. When reduced, closes and goes to the termination stage where it falls off.

What prevents elongation in both bacteria and eukaryotes?

RNA polymerase inhibitors

What is PCR (polymerase chain reaction)?

Rapid amplification of chosen segments of DNA or RNA

Specificity in RNA Polymerase-Promoter Binding

Regular subunits (5) decides what types of genes to be subscribed (σ70 or σ54.. etc.) The σ subunit of RNA polymerase determines promoter binding specificity (= which gene to be transcribed)

Error Correction by DNA Polymerases (1)

Removal of mismatched deoxynucleotide

In what directions do primers go during PCR?

Reverse primer (3' to 5') Forward primer (5' to 3')

Deoxyribonucleotides vs. Ribonucleotides

Ribo---> contains an OH at the 2' and 3' positions. Deoxyribo---> contains a H at the 2' position and an OH at the 3' position.

DNA Replicase System: Elongation

Single-stranded DNA-binding proteins (SSB) bind and stabilize separated DNA strands.

What is annealing (renaturation)?

Slow to partially annealed, then fast.

Where does the primer sequence come from?

Solid-Phase DNA synthesis

What is DNA Hybridization ("Annealing")?

Similar sequences between two species or within the same species.

DNA Replication Begins at an Origin and Usually Proceeds Bidirectionally

Steps: 1. Starts at a unique point (origin). 2. The parent strands unwind at the same time and replicate simultaneously. 3. Replication forks = dynamic points where parent DNA is being unwound and separated strands replicated. 4. Both ends of the bacterial chromosome have active replication forks (bidirectional replication).

What is a requirement of of DNA replication and the Watson-Crick Model?

Strict base-pairing rules Reminder: Watson-Crick model- each DNA strand provides the template for a new strand with a predictable complementary sequence. ---> semiconservative

Post-Translational Modifications of Histones

Such modifications (mainly occur on the tails) affect the physical properties of histones, the structure and function of the chromatin, and regulate the transcription of genes.

DNA Sequencing by Sanger Method

Template strand: ssDNA 5′ primer (synthesized) that anneals to a specific region on the template strand.

Watson-Crick base-pairing rule for the incoming NTP

The 3′-OH acts as a nucleophile, attacking the α phosphate of the incoming ribonucleoside triphosphate.

Inosinate in Anticodons

The anticodons in some tRNAs include the nucleotide inosinate (designated I). - I can form weak hydrogen bonds with A, U, and C

Protein Synthesis

The most complex biosynthetic process, protein synthesis consumes more cellular resources than any other process in most cells.

What is melting point? What factors impact melting point?

The temperature when half of the DNA is separated. 1. G-C content 2. Size 3. pH 4. ionic strength

How many tRNA molecules are there in a cell?

There are at least 32, but fewer than 61 different tRNA molecules in the cell.

______ nucleotide residues are necessary to encode each amino acid

Three

Bacterial DNA Polymerase 1

Three activities: 1. Polymerase 2. 3' to 5' exonuclease 3. 5' to 3' exonuclease Nick translation (remove and synthesize): 1. DNA repair 2. Primer removal

How many forms of DNA are there? What form is the Watson-Crick structure?

Three: 1. A form 2. B form 3. Z form B-form DNA

Under-winding and Supercoiling of Cellular DNA

To maintain an under-wound state, DNA must be in a closed circular form or bound to protein.

Transition mutation

a missense mutation in which a purine is replaced by a purine, or a pyrimidine by a pyrimidine (All three codon positions have some resistance to these mutations) A mutation in the 1st base of a codon will usually change the amino acid. The change often involves an amino acid with similar chemical properties (conservative substitution) GUU to CUU ---> Val to Leu GUU to AUU ----> Val to Ile

What does the genome contain?

all of the genes, regulatory sequences, and other DNA sequences for the cell.

Degenerate

an amino acid may be specified by more than one codon (Each codon specifies only one amino acid)

Compaction: What is DNA supercoiling?

an intrinsic property of DNA tertiary structure. • Folding of cellular DNA: - Extremely compacted - Permit access to the genetic information stored in DNA

What is phosphodiester linkage? What do 3',5'-Phosphodiester bonds do?

covalent bond that joins successive nucleotides of both DNA and RNA; link successive nucleotides formed between the 5′-phosphate group of one nucleotide unit and the 3′-hydroxyl group of the next nucleotide

Primary structure

covalent structure and nucleotide sequence

In DNA, what are A,G,T, and C symbolized as?

dA, dG, dT, and dC

In free form, deoxyribonucleotides are commonly abbreviated as?

dAMP, dGMP, dTMP, and dCMP.

RNA editing

deamination of cytidine to uridine

Topoisomers

different forms of a circular DNA that differ only in a topological property

Translational frame-shifting

insertion of residues

Contigs

long, contiguous sequences that are assembled from overlaps

Polynucleotide

longer nucleic acid

Reading Frames

method of dividing nucleotides such that a new codon begins every three nucleotide residues - Established by a specific first codon. - A new codon begins every three nucleotide residues. - No punctuation between codons (= continuous). *In principle, any given ssDNA or mRNA sequence has three possible reading frames.

Introns

non-translated DNA segments inserted between exons

Secondary structure

regular, stable structure taken up by some or all the nucleotides

Base pairs are nearly ____, ______ to the helical axis.

planar; perpendicular

Nucleic Acids

polymers of nucleotides joined by phosphodiester linkages.

What is transcription?

process by which a cellular system converts the genetic information stored in dsDNA into an RNA strand (selective). *~76% of the human genome is transcribed into RNA, most products are ncRNAs

The positive charges in histone proteins (Lys+ and Arg+) serve as what?

the counter ionic groups for the negative charges in the DNA backbone (phosphate groups).

Proteins

the end products of most information pathways (there are thousands of proteins at any given moment)

Sequencing depth

the number of times that a particular nucleotide in a genome is sequenced, on average

Translation

the overall process of mRNA-guided protein synthesis.

Transcriptome

the sum of all the RNA molecules produced in a cell under a given set of conditions ("snapshot").

Wobble Hypothesis

the third base of most codons pairs loosely with the corresponding anticodon base. The first two bases of the codon form strong WatsonCrick base pairs with the anticodon bases (=confers most of the coding specificity) When an amino acid is specified by several different codons, the codons that differ in either of the first two bases require different tRNAs. When the 1st base of the anticodon is hypoxanthine (I) (the wobble base), it can pair with U, C, or A at the 3rd base of the codon. One tRNA with the anticodon (5')ICG(3'), can pair with three different codons: CGA, CGU, and CGC * "Wobble" balances the requirements for accuracy and speed for protein synthesis.

ρ-dependent Termination in E. coli

ρ (rho) = protein factor that has an ATP-dependent RNA-DNA helicase activity 1. CA-rich sequence in the template strand, called a rut (rho utilization) element. 2. ρ protein binds to a rut site on the RNA and migrates in the 5' to 3' direction. 3. The ATP-dependent RNADNA helicase activity of r protein separates and releases the RNA.

What are the steps in Maxam-Gilbert Sequencing?

• Denature dsDNA to obtain single strand DNA (ssDNA) • Label ssDNA at the 5' end with radioactive 32P • Base-specific partial chemical modification of DNA • Cleavage of the DNA backbone at specific sites • Separate the DNA fragments by electrophoresis • Detect the DNA fragments by autoradiography.

Topo I Inhibitors as Anticancer Drugs

• Eukaryotic type I topoisomerase inhibitors: •Camptothecin: drug lead. •Camptothecin derivatives: irinotecan and topotecan are drugs used to treat colorectal and ovarian cancers. • Camptothecin and its derivatives function by trapping the Topo I-DNA complex in which the DNA is cleaved.

What are the 3 phases of DNA replication?

• Fundamental properties and mechanisms for DNA replication are essentially identical in all species: 1. Initiation 2. Elongation 3. Termination *E.coli was used as a model system because eukaryotes are more complex.

Topo II Inhibitors as Anticancer Drugs

• Human type II topoisomerase inhibitors that are anticancer drugs include doxorubicin, etoposide, and ellipticine. • These Topo II inhibitors function by stabilizing the covalent Topo-DNA (cleaved) complex (mechanism of action).

Footprinting Analysis Method

• Isolate labeled DNA fragments and separate them by electrophoresis (sequencing gel). • Sequence analysis to identify the binding site. *Protein-binding protects the specific sequence from cleavage!

Semiconservative DNA Replication

• Semiconservative: Two new DNA molecules, each has one new strand and one old strand • Established by the Meselson-Stahl experiment in 1957: - Differential density (15N vs 14N) - Time points: 1 and 2 doubling time, respectively. - Centrifugation

Next Generation Sequencing

•Miniaturization of the procedure •Massive increase in scale •Decrease in cost