BSCI 222 Genetics Chapter 12,13,14 notes
BSCI 222 Genetics Chapter 12,13,14 notes BSCI222
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This 21 page Class Notes was uploaded by Colin Fields on Monday October 17, 2016. The Class Notes belongs to BSCI222 at University of Maryland taught by Dr. Paczolt in Fall 2016. Since its upload, it has received 3 views. For similar materials see Genetics in Biology at University of Maryland.
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Date Created: 10/17/16
BSCI222 Genetics Chapter 12 DNA Replication and Recombination 1. 12.1 Genetic Information Must be Accurately Copied Every Time a Cell Divides a. Errors from replication increase exponentially so replication must make single errors per billions of base pairs to avoid deleterious effects 2. 12.2 All DNA Replication Takes Place in a Semiconservative Manner a. Semiconservative replication is so because each parent strand still exists but is paired with a new and daughter strand b. Initial models for replication were conservative, semiconservative, and dispersive c. Meselson and Stahl’s Experimen15 i. Cultured E. coli with N as the sole nitrogen source so the bases would only have this isotope ii. Split the bacteria and one portion was recultured 14 with N iii. Light and heavy DNA was separated by equilibrium density gradient centrifugation iv. Results disproved the notion of conservative replication v. Second round of culturing in N 14produced only the same two bands as before ruling our dispersive replication d. Modes of Replication i. Theta Replication 1. Circular DNA 2. Double stranded DNA unwinds and the exposed strands serve as templates for synthesis 3. This generates a loop termed a replication bubble 4. Unwinding happens at the replication fork 5. If the forks move outward in both directions there is bidirectional replication ii. Rolling Circle Replication 1. initiated by a break in a nucleotide strand that exposes a 3’-OH and a 5’-phosphate 2. Nucleotides are added to the 3’ end an displace the nucleotides of the 5’ end 3. This produces a double stranded circle and a single free strand 4. The free strand then serves as another template for replication iii. Linear Eukaryotic Replication 1. Many origins of replication 2. Two replication forks spreading outwards 3. Replicons fuse together once two forks merge iv. Requirements of Replication 1. A template consisting of single stranded DNA 2. Raw materials to be assembled into a new nucleotide strand 3. Enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule 4. Substrate contains deoxyribosenucleoside triphosphates (dNTPs) 5. Nucleotides are added to by nucleophillic attack of the 3’-OH on the 5’-phosphate of the new nucleotide, cleaving two phosphates v. Direction of Replication 1. DNA polymerases can only add to the 3’ end of a strand 2. Continuous and discontinuous replication a. Template strand exposed in the 3’-5’ direction allows continuous synthesis of a daughter strand b. Other stand is the lagging strand and is synthesized discontinuously 3. Okazaki fragments a. Short lengths of DNA produced from lagging strand 3. 12.3 Bacterial Replication Requires a Large Number of Enzymes and Proteins a. Initiation i. An initiator protein binds to an origin of replication and causes a small segment of DNA to unwind ii. This allows other single strand binding proteins to bind b. Unwinding i. DNA Helicase 1. Breaks the H-bonds between bases 2. Binds to both strands and moves in the 5’-3’ direction thus moving the replication fork ii. Single-Strand-Binding Proteins 1. Bind to any segment of single stranded DNA to prevent the formation of secondary structures 2. Tetrameric and cover 35-65 nucleotides iii. DNA Gyrase 1. A topoisomerase 2. Produces double strand breaks 3. Reduces the supercoiling thus reducing strain c. Elongation i. Synthesis of Primers 1. Primase synthesizes 10-12 nucleotide long RNAs that bind to exposed single stranded DNA and provide the 3’-OH needed by DNA polymerase to function 2. A new primer is added before ach Okazaki fragment 3. Primase forms a complex with helicase at the replication forks ii. DNA Synthesis by DNA polymerases 1. Polymerases catalyze polymerization of DNA 2. DNA Polymerase III a. 5’-3’ polymerase activity b. 3’-5’ exonuclease activity c. Adds nucleotides to the primer d. 1 subunit adds to leading strand and a second subunit adds to the lagging strand 3. DNA Polymerase I a. 5’-3’ polymerase and exonuclease activity b. 3’-5’ exonuclease activity c. Removes primers and replaces them with nucleotides 4. DNA Polymerases II, IV, V a. Mainly function in DNA repair 5. DNA Ligase a. Remaining single strand DNA break when DNA polymerase I replaces primers is repaired by Ligase b. Catalyze the formation of a phosphodiester bond without adding a nucleotide iii. Elongation at the replication fork 1. Helicase unwinds the DNA 2. Single-Strand-Binding proteins protect the exposed nucleotides 3. Gyrase removes strain ahead of the replication fork 4. Primase synthesizes primers at the beginning of each DNA fragment 5. DNA Polymerases synthesize new strands d. Termination i. Can be terminated when two replication forks meet ii. Proteins can bind to specific sequences and prevent further movement of forks e. The Fidelity of DNA Replication i. Proofreading 1. In correct pairing of the bases positions the 3’- OH out side of the active site of DNA polymerase allow its exonuclease activity time to correct the error ii. Mismatch Repair 1. Corrects errors made after replication is complete 2. Incorrect pairing creates an abnormality in secondary structure which is found by other proteins 3. Distinction between old and new DNA is made so the enzymes know which base to replace 4. 12.4 Eukaryotic DNA Replication is Similar to Bacterial plication but Differs in Several Aspects a. Eukaryotic Origins i. Autonomously replicating sequences enable any DNA to which they are attached to replicate ii. Origin recognition complexes bind to and recognize origins and unwind DNA in these regions b. The Licensing of DNA Replication i. Replication licensing factors attach to origins ii. Replication machinery then initiates replication at each licensed origin iii. The license at the origin is then lost so it cannot be a site of replication again c. Unwinding i. Essentially all the same protein types as bacteria d. Eukaryotic DNA Polymerase i. Many different types of polymerase ii. Alpha, delta, and epsilon do most of the work iii. DNA polymerase alpha 1. Primase activity 2. Also lays down 30-40 nucleotides iv. DNA polymerase delta 1. Completes replication on lagging strand v. DNA polymerase epsilon 1. Replicates the leading strand vi. Translesion DNA Polymerases 1. Have a more open active site and thus are not stalled by abnormalities in the DNA like the other polymerases 2. After the initial lesion is overcome a few more bases are laid down then the high fidelity polymerases take over again e. Nucleosome Assembly i. Original nucleosomes on parental strand are disrupted ii. Preexisting histones are redistributed to the new DNA molecules iii. Addition of newly synthesized histones to complete the nucleosomes f. The Location of Replication Within the Nucleus i. Polymerases are fixed in location and DNA is threaded through them g. DNA Synthesis and the Cell Cycle i. Rapidly dividing bacteria continually synthesize DNA ii. Eukaryotes follow checkpoints in the cell cycle 1. G1/S checkpoint holds the cell in G1 until DNA is ready to be replicated 2. After this is passed DNA is replicated in the S phase h. Replication at the Ends of Chromosomes i. The End-Replication Problem 1. Primers at the ends of chromosomes cannot be replaced with DNA because there is no 3’-OH to build from so the chromosome gradually loses information ii. Telomeres and Telomerase 1. The G overhang can be extended through the activity of telomerase 2. Telomerase contains protein and an RNA component that allows it to carry the 3’-OH with it 3. The RNA sequence pairs with the overhanging 3’ end 4. Nucleotides are added one at a time and the RNA primer gradually moves down the strand iii. Replication in Archea 1. Have similar mechanisms to eubacteria but the proteins and sequences involved are more closely related to those of eukaryotes 5. 12.5 Recombination Takes Place Through the Breakage, Alignment, and Repair of DNA Strands a. Heteroduplex DNA is where strands from pairs of homologous chromosomes associate with their complementary strand on the other of the homologous pair b. Models of Recombination i. Holliday model 1. Single strand break in each of two DNA molecules 2. Holliday juncture forms where the two strands overlap each other and reconnect with the other strand 3. The juncture then moves forward swapping the nucleotides 4. The structure can then be cleaved along the horizontal or vertical plate a. Horizontal plate cleavage produces noncrossover recombinants which don’t have any change of genes on either end of the molecule b. Vertical plate cleavage produces crossover recombinants in which genes on either end of the molecule are different than before ii. Double strand break model 1. Double strand break occurs in one of the chromosomes 2. Some nucleotides are removed from the ends of the broken strands 3. There is then strand invasion, displacement, and replication, resulting in two Holliday junctions c. Gene Conversion i. Single strand break occurs during recombination ii. Invasion occurs followed by formation of the Heteroduplex DNA iii. The nucleotides can be mismatched if they come from different alleles iv. Repair enzymes remove one set of the mismatched bases and repair the strand to match the remaining sequence v. If one strand is used as the template then gene conversion results where the true parental sequence was removed and replaced by a sequence complementary to the invading sequence BSCI222 Genetics Chapter 13 Transcription 1. 13.1 RNA, Consisting of a Single Strand of Ribonucleotides, Participates in a Variety of Cellular Functions a. An Early RNA World i. Ribozymes are catalytic RNA molecules that can cutout part of their own sequence, connect some RNA molecules, replicate others, and catalyze the formation of peptide bonds ii. RNA was likely the first biological molecule to start evolution b. The Structure of RNA i. Ribose sugar backbone which is less stable because of its free hydroxyl group ii. Uses a uracil base instead of thymine iii. Single stranded and forms secondary structures c. Classes of RNA i. Ribosomal (rRNA) which make up the subunits of ribosomes ii. Messenger (mRNA) which carries coding for proteins to ribosomes iii. Pre-messenger (pre-mRNA) which are immediate products of transcription and modified further before becoming mRNA and leaving the nucleus iv. Transfer (tRNA) which bring amino acids for specific complementary codons to ribosomes v. Small nuclear (snRNA) which form complexes with small protein subunits in the nucleus vi. Small nucleolar (snoRNA) which take part in processing of rRNA vii. Micro (miRNA) interfere with other RNAs viii. Small interfering (siRNA) interfere with other RNAs ix. Piwi-interacting (piRNA) suppress transposable elements in reproductive cells 2. Transcription is the Synthesis of an RNA Molecule from a DNA Template a. The Template i. The Transcribed Strand 1. A single strand of DNA 2. Takes place on only one of the two unwound DNA strands 3. RNA is a duplicate of the non-template strand (U for T) ii. The Transcription Unit 1. A stretch of DNA that encodes an RNA molecule and the sequences necessary for its transcription 2. Promoter sequence where the transcription apparatus binds which also determines which direction transcription takes place in and at what nucleotide it starts 3. RNA-coding region which is the stretch of the DNA that is encoded into RNA 4. Terminator sequence which signals where the transcription is to end b. The Substrate for Transcription i. Ribonucleoside triphosphates (rNTPs) are the monomers for the nucleic acid polymers ii. Nucleotides are added to the 3’-OH of the RNA molecule where two of the rNTP phosphate groups are cleaved and the remaining group participates in the phosphodiester bond c. The Transcription Apparatus i. RNA polymerase carries out all the steps of transcription ii. Bacterial RNA Polymerase 1. A single, large, multimeric enzyme 2. Sigma factor controls the binding of the polymerase to a promoter iii. Eukaryotic RNA Polymerases 1. Type I transcribes large rRNAs 2. Type II transcribes pre-mRNA and small specialized RNAs 3. Type III transcribes tRNAs, small rRNAs, and some small specialized RNAs 4. Type IV is only in plants and transcribes some siRNAs 5. Type V is only in plants and transcribes RNAs involved in heterochromatin formation 3. 13.3 Bacterial Transcription Consists of Initiation, Elongation, and Termination a. Initiation i. Promoter recognition ii. Formation of transcription bubble iii. Creation of first bonds between rNTPs iv. Escape of the transcription apparatus from the promoter v. 9-12 nucleotides are synthesized allowing transition into the elongation stage b. Elongation i. A conformational change in the polymerase after binding to the promoter makes it unable to bind to the consensus sequences of the promoter and allows it to begin transcribing downstream ii. The sigma subunit is usually released after initiation iii. The polymerase unwinds the DNA at the edge of the transcription bubble iv. Polymerase has a proofreading capability and will back up, cleave the last two nucleotides added, and begin transcription again c. Termination i. Termination usually occurs after the transcription of a terminator sequence ii. Rho-dependent terminators 1. DNA sequence causes RNA polymerase to pause 2. Upstream sequence rich in cysteine serves as a binding site for rho protein 3. Rho moves down the RNA until it reaches the polymerase where it then uses its helicase activity to unwind the RNA from the DNA iii. Rho-independent terminators 1. Contain inverted repeats which for hairpins when transcribed 2. A string of 7-9 As after the second inverted repeat which gets transcribed to uracil 3. The polyU sequence pauses the polymerase allowing the hairpins to destabilize the RNA- DNA complex which then dissociates iv. Polycistronic mRNA 1. A group of genes that were transcribed together 2. Fairly uncommon in eukaryotes 4. 13.4 Eukaryotic Transcription is Similar to Bacterial Transcription but Has Some Important Differences a. Transcription and Nucleosome Structure i. DNA is made more available and less bound to histones through the activity of many different proteins b. Promoters i. Different proteins, transcription factors, bind to different promoter regions which the recruit different RNA polymerases ii. Core Promoter 1. Located immediately upstream of a gene 2. Site to which the basal transcription apparatus binds 3. Commonly contains the TATA box sequence (TATAAA) which is located from -25 to -30 bp upstream of the start site iii. Regulatory Promoter 1. Located immediately upstream of the core promoter 2. Transcriptional activator proteins bind here iv. Polymerase I and III Promoters 1. Use a distinct set of promoters from polymerase II c. Initiation i. Assembly of RNA Polymerase II and a series of general transcription factors that for a 50 peptide complex on the promoter ii. Basal transcription apparatus consisting of RNA polymerase, a series of general transcription factors, and a complex of proteins called the mediator, is recruited to the core promoter iii. Conformational changes the cause 11-15 bp around the transcription site to separate iv. The single stranded DNA is positioned in the active site of the polymerase forming the open complex from which synthesis starts d. Elongation i. After 30 bp have been synthesized the polymerase leaves the promoter also leaving behind a number of transcription factors ii. The polymerase maintains a transcription bubble as it proceeds down the DNA e. Termination i. RNA polymerase I has a termination factor similar to rho but that binds downstream of the termination site ii. RNA polymerase III terminates after transcribing a string of uracil without the need for hairpins iii. RNA Polymerase II keeps transcribing past the end of the needed pre-mRNA. The pre-mRNA gets cleaved and a 5’-3’ exonuclease attaches to the RNA coming from the polymerase, degrades its way to the polymerase, and interacts with the polymerase ending transcription. 5. 13.5 Transcription in Archaea is More Similar to Transcription in Eukaryotes than to Transcription in Eubacteria a. Have a single RNA polymerase which is similar to the polymerases of eukaryotes b. Has some other regulators of transcription found in Eubacteria BSCI222 Genetics Chapter 14 RNA Molecules and RNA Processing 1. 14.1 Many Genes Have Complex Structures a. Gene Organization i. Not all of the information in a gene gets encoded to a protein b. Introns and Exons i. Exons are the coding regions of a gene ii. Introns are the non coding regions of a gene iii. Increase in introns is correlated to increase in organismal complexity iv. Intron Groups 1. Group I are found in eubacteria, bacteriophages, and eukaryotes and catalyze their own removal 2. Group II are found in prokaryotes and eukaryotic organelles and have a different self- splicing mechanism than group I 3. Nuclear pre-mRNA introns are located in the protein coding genes of the eukaryotic nucleus and require a number of proteins and snRNAs for splicing 4. Transfer RNA introns are found in genes for tRNA and require enzymes for splicing 2. 14.2 Messenger RNAs, Which Encode the Amino Acid Sequences of Proteins, are Modified after Transcription in Eukaryotes a. The Structure of Messenger RNA i. 5’ untranslated region which is recognized by the ribosome and precedes the start codon resulting in it not being translated ii. Protein coding region contains the codons that go into the protein and starts with the start codon and ends with an end codon iii. 3’ untranslated region which is after the stop codon and affects the stability of the mRNA b. Pre-mRNA Processing i. Prokaryotic mRNA is translated as it is transcribed so there is little opportunity for modification ii. Eukaryotic pre-mRNA is transcribed in the nucleus, modified, and then translated in the cytoplasm c. The Addition of the 5’ Cap i. A methylated nucleotide is added to the 5’ end of the pre-mRNA and methylation of one or more sugar 2’- OHs occurs ii. Stabilizes the molecule and influences intron splicing iii. Cap binding proteins attach to the cap and are were the ribosome binds before moving downstream iv. Capping is facilitated by enzymes that bind to RNA polymerase II but not I or III d. The Addition of the Poly(A) Tail i. Polyadenylation sequences occur both up and downstream of the site of 3’ cleavage ii. A large number of proteins cleave the pre-mRNA and then adenine nucleotides are added on without a template strand iii. The polyA tail confers stability to the mRNA and plays a role in recognition by the ribosome and nuclear export e. RNA Splicing i. Consensus Sequences and the Spliceosome 1. Requires 3 sequences in the intron 2. 5’ and 3’ splice sites which have short consensus sequences that indicate where the intron starts and ends 3. Branch point which is an adenine that lies upstream of the 3’ splice site 4. A large protein complex, the spliceosome, splices the intron using these three regions ii. The Process of Splicing 1. The 5’ splice site is cleaved, freeing the exon before it 2. The 5’ end of the intron folds back onto itself and attaches to the branch point forming a lariat 3. The 3’ site is the cleaved and the 3’ end of the upstream exon becomes attached to the 5’ end of the downstream exon 4. The lariat is the cleaved at the branch point and degraded by nuclear enzymes 3. 14.3 Transfer RNAs, Which Attach to Amino Acids, are Modified After Transcription in Bacterial and Eukaryotic Cells a. The Structure of tRNA i. Contain numerous modified bases ii. Bases are modified by tRNA-modifying enzymes iii. Anticodon arm contains a complementary sequence to the mRNA sequence b. tRNA Gene Structure and Processing i. Single nucleotide deletion may be done to the ends of tRNA called trimming ii. Base modifying enzymes then change some bases iii. The CCA acceptor sequence can be transcribed with the RNA or added on by enzymes later 4. 14.4 Ribosomal RNA, a Component of the Ribosome, is also Processed After Transcription a. The Structure of the Ribosome i. Large subunit ii. Small subunit iii. Protein and RNA complexes b. Ribosomal RNA Gene Structure and Processing i. Bacteria have dispersed rRNA genes while Eukaryotes have the clustered in tandem ii. Both bacteria and eukaryotes process rRNA iii. Eukaryotes use snoRNA to modify the rRNA in the nucleolus 5. 14.5 Small RNA Molecules Participate in a Variety of Functions a. RNA Interference i. Small segments of RNA are responsible for inhibiting the function of other RNAs both from the cell and from foreign RNA ii. Called the immune system of the genome b. siRNA and miRNA i. siRNA 1. Comes from mRNA, transposons, or viruses 2. Cleaved from RNA duplexes or long hairpins formed from single-stranded RNA 3. 21-25 nucleotides long 4. Degrade mRNA, inhibit transcription, and modify chromatin 5. Target genes from which they were transcribed ii. miRNA 1. Comes from a distinct gene 2. Cleaved from single-stranded RNA that forms short hairpins or double-stranded RNA 3. 21-25 nucleotides long 4. Degrade mRNA, inhibit translation, and modify chromatin 5. Target genes different from those they were transcribed from c. piRNA i. Slightly longer than other interfering RNAs and derived from single stranded RNA transcripts ii. Combine with Piwi proteins and suppress transposons in germ cells d. CRISPR RNA i. crRNA are codded by Clustered Regularly Interspaced Short Palindromic Repeats ii. Recognizes and binds to foreign DNA and marks it for degradation
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