genetics - exam 3 - summer

Types of RNA

- All synthesized from DNA template by transcription - Messenger RNA (mRNA) - Ribosomal RNA (rRNA) - Transfer RNA (tRNA) - Small nuclear RNA (snRNA) - Signal recognition particle RNA - Micro-RNA (miRNA)

The 3 Models of DNA Replication

- Conservative model: Both parental strands stay together after DNA replication - Semiconservative model: The double-stranded DNA contains one parental and one daughter strand following replication - Dispersive model: Parental and daughter DNA segments are interspersed in both strands following replication

Gene Regulation in Bacteria

- Gene (Transcription): - Genetic regulatory proteins bind to the DNA and control the rate of transcription. - In attenuation, transcription terminates soon after it has begun due to the formation of a transcriptional terminator. - mRNA (Translation): - Translational repressor proteins can bind to the mRNA and prevent translation from starting. - Riboswitches can produce an mRNA confirmation that prevents translation from starting. - Antisense RNA can bind to the mRNA and prevent translation from starting. - Protein (Posttranslation): - In feedback inhibition, the product of a metabolic pathway inhibits the first enzyme in the pathway. - Covalent modifications to the structure of a protein can alter its function.

Stages Of Translation

- Initiation: ribosomal subunits, mRNA, and tRNA assemble - Elongation: ribosome slides along mRNA and synthesizes a polypeptide - Termination: a stop codon is reached and the polypeptide is released

GATC Methylation Sites

- Involved in ensuring only one round of replication - DNA adenine methyltransferase (Dam) methylates the A on both strands several minutes after replication - Initiation of replication only occurs efficiently on fully methylated DNA so second round initiation is blocked

Open Complex

- Section of DNA that must open for transcription - TF2H unwinds DNA and phosphorylates c terminal domain

Elongation of Transcript

- The open complex formed by the action of RNA polymerase is about 17 bases long - Behind the open complex, the DNA rewinds back into a double helix - On average, the rate of RNA synthesis is about 43 nucleotides per second

Termination Models

- Allosteric: after transcribing the polyadenylation signal sequence, RNA polymerase II is destabilized and disassociates from the DNA. - Torpedo: an exonuclease binds to the 5' end of the RNA that is still being transcribed and degrades it in a 5' to 3' direction

Bacterial translation/transcription

- Bacterial translation can begin before transcription is completed - Bacteria lack a nucleus, therefore, both transcription and translation occur in the cytoplasm - As soon an mRNA strand is long enough, a ribosome will attach to its 5' end so translation begins before transcription ends - This phenomenon is termed coupling - Does not occur in eukaryotes: transcription is in nucleus and translation is in cytosol

Central Dogma

- Crick gave the name central dogma to the flow of information from DNA → RNA → protein - Transcription is the mechanism by which information encoded in the DNA template strand is copied into a complementary RNA strand - Translation uses the information encoded in the RNA copy to assemble amino acids into a polypeptide

Synthesis at the Replication Fork

- DNA pol I: removes the RNA primers and fills the resulting gap with DNA - It uses a 5' to 3' exonuclease activity to digest the RNA and 5' to 3' polymerase activity to replace it with DNA - After the gap is filled a covalent bond is still missing - DNA ligase: catalyzes the formation of a phosphodiester bond connecting the DNA fragments

Meselson and Stahl Experiment

- Grow E. coli in the presence of N15 (heavy isotope) - Switch E. coli to medium containing only N14 (light isotope) - Collect sample of cells after various times and analyze the density of the DNA by centrifugation using a CsCl gradient - Conservative should have a heavy and a light band - Semiconservative should have a half heavy/light and a light band - Dispersive should have a half heavy/light band - Meselson and Stahl's data was only consistent with the semiconservative model

Synthesis of Leading and Lagging Strands

- Leading strand: - One RNA primer is made at the origin - DNA pol III attaches nucleotides in a 5' to 3' direction as it slides toward the opening of the replication fork - Lagging strand: - Synthesis is also in the 5' to 3' direction, however it occurs away from the replication fork - Many RNA primers are required - DNA pol III uses the RNA primers to synthesize small DNA fragments (1000 to 2000 nucleotides in bacteria, 100-200 in eukaryotes) termed Okazaki fragments - The lagging strand is looped, which allows the attached DNA polymerase to synthesize the Okazaki fragments in the normal 5' to 3' direction - The polymerase synthesizing the lagging strand is also moving toward the replication fork - Upon completion of an Okazaki fragment, the enzyme releases the lagging template strand - The clamp loader complex then reloads the polymerase at the next RNA primer - Another loop is formed and the process repeats

Telomeres and DNA Replication

- Linear eukaryotic chromosomes have telomeres at both ends - The term telomere refers to the complex of telomeric DNA sequences and bound proteins - Telomeric sequences consist of moderately repetitive tandem arrays (3' overhang that is 12-16 nucleotides long) - Telomeric sequences typically consist of several guanine nucleotides and many thymine nucleotides - DNA polymerases synthesize DNA only in the 5' to 3' direction and cannot initiate DNA synthesis, so at the 3' ends of linear chromosomes - the end of the strand cannot be replicated - The cell solves this problem by adding DNA sequences to the ends of telomeres, which requires a specialized mechanism catalyzed by the enzyme telomerase - Telomerase contains protein and RNA: the RNA is complementary to the DNA sequence found in the telomeric repeat which allows the telomerase to bind to the 3' overhang

Eukaryotic Pre-mRNA

- Most mature mRNAs have a 7-methylguanosine covalently attached at their 5' end, known as capping - Capping occurs as the pre-mRNA is being synthesized by RNA pol II, usually when the transcript is only 20 to 25 bases long - The 7-methylguanosine cap structure is recognized by cap-binding proteins - Cap-binding proteins play roles in the movement of some RNAs into the cytoplasm, early stages of translation, and splicing of introns

Polyadenylation

- Most mature mRNAs have a string of adenine nucleotides at their 3' ends, called the polyA tail - The polyA tail is not encoded in the gene sequence, it is added enzymatically after the gene is completely transcribed

RNA transcripts and their different functions

- Structural genes: Transcribed in to mRNA and constitute about 90% of all genes - Non Structural genes are not translated: Ribosomes, Spliceosomes, Signal Recognition Particle, Telomerase

The Genetic Code

- The nucleotide information that specifies the amino acid sequence of a polypeptide is called the genetic code - To code for 20 different amino acids, the four bases in an mRNA (A, U, G, and C) are used in combinations of three - Each three-letter word (triplet) of the code is called a codon - Three-letter codons in DNA are transcribed into complementary three-letter RNA codons - Francis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order - Introduced single nulcleotide insertions or deletions and looked for mutations - Frameshift mutations - Indicate importance of reading frame - Marshall Nirenberg identified the codons that specify each amino acid - Stop codons: 3 codons (UAA, UGA, UAG) used to terminate translation, do not code for amino acids - Start codon: Codon (AUG) (specifying methionine) is always the first codon read in an mRNA translation - Code is degenerate, meaning that some amino acids are specified by more than one codon - only two amino acids, methionine and tryptophan, are specified by a single codon - The four mRNA nucleotides combine to form 64 different 3-letter combinations (codons) - 61 sense codons specify amino acids - The genetic code is commaless, with no indicators to mark the end of one codon and the beginning of the next - The genetic code is universal - essentially the same in all living organisms and viruses - Selenocysteine and pyrrolysine are sometimes called the 21st and 22nd amino acids - Found in specialty enzymes - Encoded by UGA and UAG codons, respectively - Attached by tRNAs that carry them to the ribosome - Codon and downstream sequences in mRNA are needed to incorporate these amino acids

Transcription Apparatus

- Three categories of proteins are required for basal transcription to occur at the promoter: RNA polymerase II, Five different proteins called general transcription factors (GTFs), and A protein complex called mediator - Basal transcription apparatus (RNA pol II + the five GTFs) - The third component required for transcription is a large protein complex termed mediator - It mediates interactions between RNA pol II and various regulatory transcription factors that bind enhancers or silencers - It's subunit composition is complex and variable - Core subunits partially wraps around RNA pol II - Mediator may phosphorylate the CTD of RNA polymerase II and it may regulate the ability of TFIIH to phosphorylate the CTD, therefore it plays a pivotal role in the switch between transcriptional initiation and elongation

Numbering system of promoters

- Transcription start site is +1 - TATA box is -10 sequence - Promoter is between -10 and -35 sequences - Up mutations move towards consensus sequence and speed up transcription

RNA Processing

- Transfer RNAs are also made as large precursors - These have to be cleaved at both the 5' and 3' ends to produce mature, functional tRNAs - Cleaved by exonuclease and endonuclease - exonucleases: cleave a covalent bond between two nucleotides at one end of a strand (exo = end) - endonucleases: can cleave bonds within a strand (endo = not end)

Pre-mRNA Splicing

- In eukaryotes, the transcription of structural genes produces a long transcript known as a pre-mRNA - This RNA is altered by splicing and other modifications, before it leaves the nucleus - Splicing requires the aid of a multicomponent structure known as the spliceosome - It is composed of several subunits known as snRNPs (pronounced "snurps") - Each snRNP contains small nuclear RNA and a set of proteins - The subunits of a spliceosome carry out several functions: - Bind to an intron sequence and precisely recognize the intron-exon boundaries - Hold the pre-mRNA in the correct configuration - Catalyze the chemical reactions that remove introns and covalently link exons

DNA Polymerases (eukaryotes)

- Mammalian cells contain well over a dozen different DNA polymerases - Four: alpha (α), delta (δ), epsilon (ε) and gamma (γ) have the primary function of replicating DNA - α, δ and ε = Nuclear DNA - γ = Mitochondrial DNA - DNA pol a is the only polymerase to associate with primase - The DNA pol α/primase complex synthesizes a short RNA-DNA hybrid primer (10 RNA nucleotides followed by 20 to 30 DNA nucleotides) - The exchange of DNA pol α for ε or δ is required for elongation of the leading and lagging strands (polymerase switch) - DNA pol ε is used for the processive elongation of the leading strand - DNA pol δ is used for the lagging strands - DNA polymerases also play a role in DNA repair: DNA pol β plays a role in removal of incorrect bases from damaged DNA - Other DNA polymerases are translesion-replicating polymerases involved in the replication of damaged DNA - They can synthesize a complementary strand over the abnormal region

Beadle and Tatum

- Set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses - Studied Neurospora crassa (bread mold) - Used X-rays to damage DNA - looked for fungal cells lacking specific enzymes required for the biochemical pathway producing the amino acid arginine - One-gene/one-enzyme hypothesis was modified to one-gene/one-polypeptide hypothesis

Synthesis of RNA Primers and DNA

- Short (10 to 12 nucleotides) RNA primers are synthesized by primase - These short RNA strands start, or prime, DNA synthesis - The leading strand has a single primer, but the lagging strand needs multiple primers - They are eventually removed and replaced with DNA - DNA polymerase enzymes are responsible for synthesizing the DNA

Effector Molecules

- Small effector molecules affect transcription regulation - These bind to regulatory proteins but not to DNA directly - A small effector molecule may increase transcription - These molecules are termed inducers: • Bind to activators and cause them to bind to DNA - Bind to repressors and prevent them from binding to DNA - Genes that are regulated in this manner are termed inducible - A small effector molecule may inhibit transcription - Corepressors bind to repressors and cause them to bind to DNA - Inhibitors bind to activators and prevent them from binding to DNA - Genes that are regulated in this manner are termed repressible

Srb and Horowitz

- grew in MM (minimal medium) with and without ornithine and citrulline intermediates involved in the arginine biosynthesis pathway - the biosynthesis of arginine occurs in a series of steps, with each step controlled by a gene that encodes the enzyme for the step

RNA Editing

- Change in the nucleotide sequence of an RNA can involve addition or deletion of particular bases, and can also occur through conversion of a base - Deamination converts RNA nucleotides to new forms - First discovered in trypanosomes - Now known to occur in many organisms

Accuracy of Replication

- DNA polymerases catalyzes the formation of a covalent (ester) bond between the innermost phosphate group of the incoming deoxyribonucleoside triphosphate and the 3'-OH of the sugar of the previous deoxynucleotide - In the process, the last two phosphates of the incoming nucleotide are released in the form of pyrophosphate (PPi)

origin of Chromosomal replication (oriC)

- DnaA proteins bind to DnaA boxes and to each other - Additional proteins bind to bend DNA - Strands separate at AT-rich region - DnaB/helicase travels along the DNA in the 5' to 3' direction using energy from ATP to unwind DNA

Translation Initiation Eukaryotes

- In eukaryotes, the assembly of the initiation complex is similar to that in bacteria, however, additional factors are required - eukaryotic Initiation Factors are denoted eIF - The initiator tRNA is designated tRNAmet and carries a methionine rather than a formylmethionine - The start codon for eukaryotic translation is AUG. - Ribosome scans from the 5' end of mRNA until it finds the AUG start codon (not all AUGs can act as a start) - Rules for optimal translation initiation are called Kozak's rules - The start codon for eukaryotic translation is usually the first AUG after the 5' Cap - An initiation factor protein complex (eIF4) binds to the 5' cap in mRNA - These are joined by a complex consisting of the 40S subunit, tRNAmet, and other initiation factors - The entire assembly moves along the mRNA scanning for the right start codon - Once it finds this AUG, the 40S subunit binds to it and the 60S subunit joins, creating the 80S initiation complex

Amino Acids

- Nonpolar are hydrophobic, often buried within the interior of a folded protein - Polar and charged amino acids are hydrophilic, more likely to be on the surface of a protein

RNA POL types

- Nuclear DNA is transcribed by three different RNA polymerases: - RNA pol I: Transcribes all rRNA genes (except for the 5S rRNA) - RNA pol II: Transcribes all protein-encoding (structural) gene (all mRNAs), and some snRNA genes needed for splicing - RNA pol III: Transcribes all tRNA genes, the 5S rRNA gene, and microRNA genes

Levels of Structure in Proteins

- Primary: amino acid sequence - Secondary: alpha helix or beta sheet - Tertiary: 3D structure - Quaternary: 2 or more polypeptides

Alternative splicing

- Allows different polypeptides to be made from the same gene - A pre-mRNA with multiple introns can be spliced in different ways - This will generate mature mRNAs with different combinations of exons - Two (or more) polypeptides can be derived from a single gene - This allows an organism to carry fewer genes in its genome - This variation in splicing can occur in different cell types or during different stages of development - Degree of splicing and alternative splicing varies greatly among different species: - Baker's yeast: ~ 6300 genes, ~ 300 encode premRNAs that are spliced - only a few have been shown to be alternatively spliced - Humans: ~ 22,000 protein-encoding genes, most contain one or more introns - Estimates suggest ~ 70% are alternatively spliced - Constitutive exons are always found in the mature mRNA from all cell types and encode polypeptide segments that are necessary for its general structure and function - Other exons, called alternative exons vary from one cell type to another and subtly change the function of the protein to meet the needs of the cell type in which it is found - Regulated by splicing factors such as SR proteins

lac Operon

- An operon is a regulatory unit consisting of a few structural genes under the control of one promoter - An operon encodes a polycistronic mRNA that contains the coding sequence for two or more structural genes - This allows a bacterium to coordinately regulate a group of genes that encode proteins with a common functional goal - An operon contains several important DNA sequences: Promoter; terminator; structural genes; operator - The actual lac operon: - DNA elements: - Promoter: Binds RNA polymerase - Operator: Binds the lac repressor protein - CAP site: Binds the Catabolite Activator Protein (CAP) - Structural genes: - lacZ: Encodes β-galactosidase - Enzymatically cleaves lactose and lactose analogues and converts lactose to allolactose (an isomer) - lacY: Encodes lactose permease - Membrane protein required for transport of lactose and analogues - lacA: Encodes galactoside transacetylase - Covalently modifies lactose and analogues - The lacI gene: - Not considered part of the lac operon - Has its own promoter, the i promoter - Constitutively expressed at fairly low levels - Encodes the lac repressor - The lac repressor protein functions as a tetramer - Only a small amount of protein is needed to repress the lac operon

Ribosome Function

- Checks codon/anticodon interaction - Translocate 3 nucleotides at a time - Carries peptidyl transferase - Function in the rRNA of the large subunit

Proteins in Bacterial DNA Replication

- DNA Helicase: breaks the hydrogen bonds between the DNA strands. - Topoisomerase II: alleviates positive supercoiling. - Single-stranded binding proteins: keep the parental strands apart - Primase: synthesizes an RNA primer - DNA polymerase III: synthesizes a daughter strand of DNA - DNA polymerase I: excises the RNA primers and fills in with DNA (not shown) - DNA ligase: covalently links the Okazaki fragments together

Replication Complexes

- DNA helicase and primase are physically bound to each other to form a complex called the primosome - This complex better coordinates the actions of helicase and primase - The primosome is physically associated with two DNA polymerase holoenzymes to form the replisome - Two DNA pol III proteins act in concert to replicate both the leading and lagging strands - The two proteins form a dimeric DNA polymerase that moves as a unit toward the replication fork - DNA polymerases can only synthesize DNA in the 5' to 3' direction, so synthesis of the leading strand is continuous and the lagging strand is discontinuous

Unwinding of the Helix

- DNA helicase separates the two DNA strands by breaking the hydrogen bonds between them - This generates positive supercoiling ahead of each replication fork - Topoisomerase II (DNA gyrase) travels ahead of the helicase and alleviates these supercoils - Single-strand binding proteins bind to the separated DNA strands to keep them apart - Bases are exposed and can hydrogen bond with individual nucleotides

DNA Polymerases (in E.coli)

- DNA pol II, IV and V: DNA repair and replication of damaged DNA - DNA pol III: Responsible for most of the DNA replication - DNA pol III holoenzyme: complex of all 10 subunits - α subunit: Synthesizes DNA - ε subunit: 3' to 5' proofreading (removes mismatched nucleotides) - θ subunit: Accessory protein that stimulates the proofreading function - β subunit: Clamp protein, which allows DNA polymerase to slide along the DNA without falling off - τ, γ, δ, δ′, ψ, and χ subunits: Clamp loader complex, involved with helping the clamp protein bind to the DNA - DNA pol I: Composed of a single polypeptide that removes the RNA primers and replaces them with DNA - Structure resembles a human right hand: Template DNA is threaded through the palm; thumb and fingers wrapped around the DNA - DNA polymerases cannot initiate DNA synthesis by linking two individual nucleotides (fixed by RNA primers) - DNA polymerases can attach nucleotides only in the 5' to 3' direction, but the two strands are anti-parallel and go in opposite directions (synthesizing the new strands both toward, and away from, the replication fork)

Processive Enzyme

- DNA polymerase III remains attached to the template as it is synthesizing the daughter strand - This processive feature is due to several different subunits in the DNA pol III holoenzyme: - β subunit (clamp protein) forms a dimer in the shape of a ring around template DNA, and once bound, the β subunits can freely slide along dsDNA - Complex of several subunits functions as a clamp loader - In the absence of the β subunit, DNA pol III falls off the DNA template after about 10 nucleotides have been polymerized (Its rate is ~ 20 nucleotides per second) - In the presence of the β subunit, DNA pol III stays on the DNA template long enough to polymerize up to 500,000 nucleotides (Its rate is ~ 750 nucleotides per second)

Fidelity Mechanisms

- DNA replication exhibits a high degree of fidelity: mistakes during the process are extremely rare - DNA pol III makes only one mistake per 108 bases made - Stability of proper base pairs: - Complementary base pairs have much higher stability than mismatched pairs - Stability of base pairs only accounts for part of the fidelity (error rate for mismatched base pairs is 1 per 1,000 nucleotides) - Configuration of the DNA polymerase active site: - Helix distortion caused by mispairing prevents incorrect nucleotide fitting properly in active site - This induced-fit phenomenon decreases the error rate to a range of 1 in 100,000 to 1 million - Proofreading function of DNA polymerase: - DNA polymerases can identify a mismatched nucleotide and remove it from the daughter strand - The enzyme uses a 3' to 5' exonuclease activity to digest the newly made strand until the mismatched nucleotide is removed - DNA synthesis then resumes in the 5' to 3' direction

Nirenberg and Khorana

- Deciphered the genetic code - Khorana developed a novel method to synthesize RNA - They first created short RNAs (2 to 4 nucleotides long) that had a defined sequence - These were then linked together enzymatically to create long copolymers - They used these copolymers in a cell-free translation system: synthetic RNA "UC" forms polymer with codon possibilities UCU, CUC - Nirenberg and Leder discovered that a 3 nucleotide RNA could cause a ribosome to bind a tRNA - They mixed one triplet in 20 tubes each with a different radiolabeled amino acid - Only amino acids stuck to the ribosomes were retained - The tube with a large amount of retained radioactivity was the corresponding amino acid

tRNA and mRNA

- During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA - tRNAs are named according to the amino acid they bear - The anticodon is anti-parallel to the codon - The enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases - There are 20 types, one for each amino acid - Aminoacyl-tRNA synthetases catalyze a two-step reaction involving three different molecules: Amino acid, tRNA and ATP, resulting in a charged tRNA - The aminoacyl-tRNA synthetases are responsible for the "second genetic code" - The selection of the correct amino acid must be highly accurate or the polypeptides may be nonfunctional - Error rate is less than one in every 10,000 - Sequences throughout the tRNA including but not limited to the anticodon are used as recognition sites - Modified bases may affect translation rates, recognition by aminoacyl-tRNA synthetases, and codon-anticodon recognition

Translation Elongation

- During this stage, amino acids are added to the polypeptide chain, one at a time - In bacteria: 15-20 amino acids per second - In eukaryotes: 2-6 amino acids per second - After peptide bond formation, tRNAs at the P and A sites move into the E and P sites - The 23S rRNA (a component of the large subunit) is the actual peptidyl transferase, thus, the ribosome is a ribozyme

Multiple Origins of Replication

- Eukaryotes have long linear chromosomes and require multiple origins of replication to ensure that the DNA can be replicated in a reasonable amount of time - In 1968, Huberman and Riggs provided evidence for multiple origins of replication - DNA replication proceeds bidirectionally from many origins of replication - Origins of replication in Saccharomyces cerevisiae (yeast) are termed ARS elements (Autonomously Replicating Sequence) - They are about 50 bp in length and have a high percentage of A and T because there are less H bonds so it is easier to break - ARS consensus sequence (ACS): ATTTAT(A or G)TTTA - Origins in more complex eukaryotes are not fully defined, many occur at sites defined by chromatin structure, not sequence - Replication begins with assembly of the prereplication complex (preRC) - Includes the Origin recognition complex (ORC), a six-subunit complex that acts as the first initiator of eukaryotic DNA replication - Other preRC proteins include MCM Helicase - Binding of MCM completes DNA replication licensing - These origins are able to begin DNA synthesis

Eukaryotic DNA Replication

- Eukaryotic DNA replication is not as well understood as bacterial replication - The two processes do have extensive similarities, bacterial enzymes have also been found in eukaryotes - Nevertheless, DNA replication in eukaryotes is more complex because of large linear chromosomes, chromatin tightly packed within nucleosomes, and more complicated cell cycle regulation

Shine-Dalgarno sequence

- In bacteria the binding of mRNA to the 30S subunit is facilitated by a ribosomal-binding site or Shine-Dalgarno sequence - This is complementary to a sequence in the 16S rRNA - Interact via hydrogen bonds - The mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex, requiring three Initiation Factors - The initiator tRNA recognizes the start codon in mRNA - In bacteria, this tRNA is designated tRNAfmet: - It carries a methionine that has been covalently modified to N-formylmethionine - The start codon is AUG, but in some cases GUG or UUG - In all three cases, the first amino acid is N-formylmethionine

Binding sites

- In the absence of the inducer, this repressor protein blocks transcription - The presence of the inducer causes a conformational change that inhibits the ability of the repressor protein to bind to the DNA and transcription proceeds. - This activator protein cannot bind to the DNA unless an inducer is present. - When the inducer is bound to the activator protein, this enables the activator protein to bind to the DNA and activate transcription. - In the absence of a corepressor, this repressor protein will not bind to the DNA, therefore, transcription can occur. - When the corepressor is bound to the repressor protein, a conformational change occurs that allows the repressor to bind to the DNA and inhibit transcription. - This activator protein will bind to the DNA without the aid of an effector molecule. - The presence of an inhibitor causes a conformational change that inhibits the ability of the activator protein to bind to the DNA inhibiting transcription.

Transcription

- Occurs in three stages: Initiation, Elongation, and Termination - These steps involve protein-DNA interactions - Proteins such as RNA polymerase interact with DNA sequences

Termination of Replication

- On the opposite side of the chromosome to oriC is a pair of termination sequences called ter sequences - T1 stops counterclockwise forks, T2 stops clockwise forks - The protein tus (termination utilization substance) binds to the ter sequences - tus bound to the ter sequences stops the movement of the replication forks - DNA replication ends when oppositely advancing forks meet (usually at T1 or T2) - Finally DNA ligase covalently links the two daughter strands - DNA replication often results in two intertwined molecules called catenanes, which are separated by the action of topoisomerase

Jacob, Monod, and Pardee

- One type of mutant involved a defect in the lacI gene (designated lacI-) - It resulted in the constitutive expression of the lac operon even in the absence of lactose - The lacI- mutations mapped very close to the lac operon - Hypothesis: the lacI- mutation either results in the synthesis of an internal inducer or eliminates the function of a lac repressor that can diffuse throughout the cell - They used bacterial conjugation methods to introduce different portions of the lac operon into different strains - They identified F' factors (plasmids) that carried portions of the lac operon (an F' factor that carries the lacI gene) - Bacteria that receive this will have two copies of the lacI gene, one on the chromosome and the other on the F' factor - These are called merozygotes, or partial diploids - The two lacI genes in a merozygote may be different alleles: lacI- on the chromosome and lacI+ on the F' factor - Genes on the F' factor are not physically connected to those on the bacterial chromosome - If hypothesis 1 is correct : The inducer protein produced from the chromosome can diffuse and activate the lac operon on the F' factor - If hypothesis 2 is correct : The repressor from the F' factor can diffuse and turn off the lac operon on the bacterial chromosome

Template strand, Nontemplate strand and coding strand

- Opposite strand is called the non-template or coding strand or sense strand - The base sequence is identical to transcript Template strand is called anti-sense

Flap Endonuclease

- Polymerase δ runs into primer of adjacent Okazaki fragment, and pushes a portion of primer into short flap - Flap endonuclease removes the primer - Long flaps are removed by DNA2 nuclease/helicase, which cuts long flap into short flap

RNA Transcript Modification

- Processing of rRNA and tRNA transcripts to smaller functional pieces - 5' Capping and 3' polyA tailing of mRNA transcripts - Many nonstructural genes are initially transcribed as a large RNA - This large RNA transcript is enzymatically cleaved into smaller functional pieces

Bacterial RNA Polymerase

- RNA polymerase is the enzyme that catalyzes the synthesis of RNA - In E. coli, the RNA polymerase holoenzyme is composed of: - Core enzyme: Five subunits = α2ββ'ω - α2: keeps complex together - ββ': enzyme catalysis - Sigma factor: One subunit = σ - σ: reorganizes promoter sequence

Garrod

- Recognized that alkaptonuria is inherited via a recessive allele - Proposed that patients with the disease lacked a particular enzyme - These ideas connected genes to enzymes - He described the disease as an inborn error of metabolism

Factors Affecting Transcription

- Regulatory elements are short DNA sequences that affect the binding of RNA polymerase to the promoter - Transcription factors (proteins) bind to these elements and influence the rate of transcription - There are two types of regulatory elements: - Enhancers: Stimulate transcription - Silencers: Inhibit transcription - They vary widely in their locations but are often found in the -50 to -100 region - Factors that control gene expression can be divided into two types, based on their "location": - cis-acting elements: DNA sequences that exert their effect only over a particular gene (TATA box, enhancers and silencers) - trans-acting elements: Regulatory proteins that bind to such DNA sequences

DNA Replication

- Relies on the complementarity of DNA strands (A pairs with T and G pairs with C) - The two complementary DNA strands (parental strands) come apart and serve as a template strand for the synthesis of new complementary DNA strands (daughter strands)

Splicing Mechanisms

- Splicing involves removal of the intron RNA and linkage of the exon RNA by a phosphodiester bond - Splicing among group I and II introns is termed self-splicing: - Splicing does not require the aid of enzymes, instead the RNA itself functions as its own ribozyme - Group I and II self-splicing can occur in vitro without the additional proteins, however, in vivo, proteins known as maturases often enhance the rate of splicing - Group I splicing involves binding of a free guanosine to a site within the intron- leads to cleavage at 3' end of exon 1 - Then bond between different nucleotide in the intron and the 5' end of exon 2 is cleaved - The 3' end of exon 1 then forms a covalent bond with the 5' end of exon - Group II self splicing is similar except that the 2' OH group in adenosine begins catalytic process. - Pre-mRNA splicing requires the aid of structure known as spliceosome

Telomere Length and Cancer

- Telomeres tend to shorten in actively dividing cells from 8,000 bp at birth to 1,500 in elderly person - Cells become senescent (aged) when telomeres are short and lose their ability to divide - Insertion of highly active telomerase can block senescence - Cancer cells commonly carry mutations increasing activity of telomerase, which prevents telomere shortening and senescence - May be a target for anti-cancer drug treatments

Termination of Bacterial transcription

- Termination is the end of RNA synthesis and occurs when the short RNA-DNA hybrid of the open complex is forced to separate - This releases the newly made RNA as well as the RNA polymerase - E. coli has two different mechanisms for termination: - rho-dependent termination: requires a protein known as ρ (rho) - rho-independent termination: does not require ρ - ρ-independent termination is facilitated by two sequences in the RNA: - A uracil-rich sequence located at the 3' end of the RNA - A stem-loop structure upstream of the uracil-rich sequence

DNA and RNA Nucleotides

- The DNA "alphabet" consists of four letters representing the four bases of DNA nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C) - The RNA "alphabet" consists of A, U, G, and C - the base uracil (U) acts in place of thymine (T) - The sequence of RNA nucleotides in mRNA is translated into a polypeptide containing 20 different types of amino acids

Initiation of Transcription in Bacteria

- The RNA polymerase holoenzyme binds loosely to the DNA, then scans along the DNA, until it encounters a promoter region - When it does, the sigma factor recognizes both the -35 and -10 regions - A region within the sigma factor that contains a helix-turn-helix structure is involved in a tighter binding to the DNA - The binding of the RNA polymerase to the promoter forms the closed complex - The open complex is formed when the TATAAT box in the -10 region is unwound (A-T bonds are more easily separated) - A short RNA strand is made within the open complex and the sigma factor is released marking the end of initiation - The core enzyme (α2ββ'ω) now slides down the DNA to synthesize an RNA strand (elongation phase)

Translation Termination

- The final stage occurs when a stop codon is reached in the mRNA - In most species there are three stop or nonsense codons: UAG, UAA, UGA - These codons are not recognized by tRNAs, but by proteins called release factors (the 3-D structure tRNAs) - Bacteria have three release factors: - RF1 recognizes UAA and UAG - RF2 recognizes UAA and UGA - RF3 does not recognize any of the three codons - required for the termination process - Eukaryotes only have two release factors: - eRF1 recognizes all three stop codons - eRF3 required for termination process

Bacterial DNA Replication

- The formation of two replication forks at the origin of replication - DNA synthesis begins at a site termed the origin of replication - Each bacterial chromosome has only one origin of replication - Synthesis of DNA proceeds bidirectionally around the bacterial chromosome - The two replication forks eventually meet at the opposite side of the bacterial chromosome ending replication

Isolation of Mutants

- The isolation of mutants has been crucial in elucidating DNA replication (discovery of DNA pol I and other enzymes involved in DNA synthesis) - DNA replication is vital for cell division, so most mutations that block DNA synthesis are lethal - A type of conditional mutant is a temperature-sensitive (ts) mutant, which can survive at the permissive temperature, but will fail to grow at the nonpermissive temperature - E. coli has many vital genes that are not involved in DNA replication, so only a subset of ts mutants would carry mutations affecting the replication process, therefore, researchers in the 1960s had to screen thousands of ts mutants to get to those involved in DNA replication ("brute force" genetic screen) - The dna mutants fell into two groups when shifted to the non-permissive temperature: - Some showed a rapid arrest, meaning these genes encoded enzymes needed for replication of the DNA - Other mutants completed their current round of replication but could not start another, meaning encoded genes needed for initiation of replication

Mechanism of induction of lac operon

- The lac operon can be transcriptionally regulated by a repressor protein and by an activator protein - The first method is an inducible, negative control mechanism involving the lac repressor protein - The inducer is allolactose which binds to the lac repressor and inactivates it - In the absence of the inducer allolactose, the repressor protein is tightly bound to the operator site, thereby inhibiting the ability of RNA polymerase to transcribe the operon. - When allolactose is available, it binds to the repressor. - This alters the conformation of the repressor protein, which prevents it from binding to the operator site. - Therefore, RNA polymerase can transcribe the operon.

Transcriptional Regulation

- The most common way to regulate gene expression in bacteria is by influencing the initiation of transcription • The rate of RNA synthesis can be increased or decreased - Transcriptional regulation involves the actions of two main types of regulatory proteins - Repressors: Bind to DNA and inhibit transcription - Activators: Bind to DNA and increase transcription - Negative control refers to transcriptional regulation by repressor proteins - Positive control refers to regulation by activator proteins

Ribosomes

- Translation occurs on the surface of a large macromolecular complex termed the ribosome - Bacterial cells have one type of ribosome found in their cytoplasm - Eukaryotic cells have two types of ribosomes: one type is found in the cytoplasm, the other is found in mitochondria or chloroplasts - Ribosomes have a small subunit (30S) and a large subunit (50S) - During bacterial translation, the mRNA lies on the surface of the 30S subunit - As a polypeptide is being synthesized, it exits through a channel within the 50S subunit - Ribosomes contain three discrete sites: Peptidyl site (P site), Aminoacyl site (A site), and Exit site (E site)

The cycle of lac operon induction and repression

- When lactose becomes available, a small amount of it is taken up and converted to allolactose by β-galactosidase. - The allolactose binds to the repressor, causing it to fall off the operator site. - lac operon proteins are synthesized. promoting the efficient uptake and metabolism of lactose. - The lactose is depleted and Allolactose levels decrease. - Allolactose is released from the repressor, allowing it to bind to the operator site. - Most proteins involved with lactose utilization are degraded.

Wobble Rule

- With the exception of serine, arginine and leucine, codon degeneracy always occurs at the codon's third position - Crick proposed the wobble hypothesis: In the codon-anticodon recognition process, the first two positions pair strictly according to the A - U /G - C rule, however, the third position can actually "wobble" or move a bit thus tolerating certain types of mismatches - tRNAs that are able to recognize the same codon are termed isoacceptor tRNAs