Molecular Biology/Genetics. DNA replication RNA transcription Protein synthesis Mutations Biotechnology Regulation of gene expression

Molecular Biology/Genetics DNA replication RNA transcription Protein synthesis Mutations Biotechnology Regulation of gene expression

Nucleic acids are polymers, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. DNA stores genetic information. The DNA is use to make RNA, and RNA assembles amino acids into protein. DNA trna rrna

DNA vs Protein Proteins

USABO2011, B or C

Nucleotides (monomers) Nucleic acids are polymers, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. DNA OH RNA Nitrogenous base DNA RNA cytosine cytosine adenine adenine guanine guanine thymine uracil Pentose sugar DNA RNA deoxyribose ribose Each nucleotide has three components: a nitrogenous base, a 5-carbon sugar, and a phosphate group. If the sugar is deoxyribose the polymer is DNA. If the sugar is ribose, the polymer is RNA. When all three components are combined, they form a nucleic acid. Base DNA cytosine, adenine, guanine, thymine RNA cytosine, adenine, guanine, uracil Sugar deoxyribose ribose Phosphate same same

Nitrogenous base (50% purines and 50% pyrimidines in DNA or RNA) A G T C U

G=32% So C=32% So A=18% and T=18% USABO2012, 42C

DNA RNA

UK(BBO)2015,11C

The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the random coil or single-stranded (ssdna) state. Tm depends on: 1. the length of the DNA molecule 2. its specific nucleotide sequence. i.e. GC contents (%GC). 3. PH 4. Ionic strength

USABO2013, 6C

rrna

DNA replication in eukaryotes

DNA replication DNA replication is the process of producing two identical replicas from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of two strands and each strand of the original DNA molecule serves as a template for the production of the complementary strand, a process referred to as semiconservative replication. Initial DNA replication begins at specific site called origins of replication. 1. Helicase unwinds the helix into two strands. 2. Primase synthesizes RNA primers, using the parental DNA as a template. The RNA primer binds to the template DNA.

DNA replication 1. Helicase unwinds the DNA, producing a replication fork. Topoisomerase removes twists and knots that form doublestranded template as a result of the unwinding induced by helicase. 2. Primase initiates DNA replication at special nucleotide sequences (origins of replication) with RNA primers. Primase synthesizes RNA primers, using the parental DNA as the template. 3. DNA polymerase III attaches to the RNA primers and begins elongation from 5 to 3, the adding of DNA nucleotides to the complementary strand. 4. The leading complementary stand is assembled continuously toward the replication fork as the double-helix DNA uncoils. 5. The lagging complementary strand is away from the replication fork in multiple, short Okazaki fragments. Each new Okazaki fragment begins when DNA polymerase attaches to an RNA primer. 6. The Okazaki fragments are joined by DNA ligase. 7. The RNA primers are replaced with DNA nucleotides by DNA polymerase I. Chromosome structure Origins of replications Prokaryotes circular Unique /one Eukaryotes Linear multiple

DNA repair: three mechanisms are used to repair DNA replication errors 1. Proofreading by DNA polymerase. 2. Mismatch repair protein to errors that escape the proofreading. 3. Excision repair proteins identify and remove damaged nucleotides.

1D. 2B. 3D. 4D.5E

Protein Synthesis The process that describes how enzymes and other proteins are made from DNA is called protein synthesis (translation). Three kinds of RNA molecules: Messenger RNA (mrna) Transfer RNA (trna) Ribosomal RNA (rrna) Three steps in protein synthesis: 1. Transcription (in nucleus) 2. RNA processing (in nucleus) 3. Translation (in cytoplasm)

Transcription Transcription is the first step of gene expression, in which a particular segment of DNA is copied into mrna by the enzyme RNA polymerase. mrna copies the information stored in DNA and carries it to the ribosome Transcription proceeds in the following general steps: 1. Initiation: a. One or more transcription factor protein binds to the RNA polymerase holoenzyme, allowing it to bind to promoter DNA. b. RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides. 2. Elongation a. RNA polymerase adds matching RNA nucleotides to the complementary nucleotides of one DNA strand in the 5 to 3 direction. b. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand. 3. Termination a. Termination occurs when the RNA polymerase reaches the termination signal (stop codons). b. Hydrogen bonds of the untwisted RNA-DNA helix break, freeing the newly synthesized RNA strand. Hydrogen bonds of the RNA- DNA helix : a. Guanines and cytosines pair up. b. Adenines and uracils pair up. Transcription proceeds

Transcription factor protein binds to the RNA polymerase to form a protein complex, allowing it to bind to promoter DNA

RNA processing: Eukaryotic cells modify RNA after transcription: 1. Alteration of mrna Ends, 2. Split Genes and RNA Splicing. 1. Alteration of mrna Ends: Each end of a pre-mrna molecule is modified in a particular way. a. The 5 end is synthesized first; it receives a 5 -cap, a modified form of a guanine (G) nucleotide added onto the 5 end after transcription of the first 20 40 nucleotides. b. At the 3 end, an enzyme adds 50 250 more adenine (A) nucleotides, forming a poly-a tail. The 5 cap and poly-a tail share several important functions. First, they seem to facilitate the export of the mature mrna from the nucleus. Second, they help protect the mrna from degradation by hydrolytic enzymes. And third, they help ribosomes attach to the 5 end of the mrna once the mrna reaches the cytoplasm. 1. Alteration of mrna Ends 2. Split RNA Splicing

RNA processing: Eukaryotic cells modify RNA after transcription: 1. Alteration of mrna Ends. 2. RNA Splicing. 2. RNA Splicing. DNA nucleotides that codes for a eukaryotic polypeptide is usually not continuous; it is split into segments. The noncoding segments of nucleic acid that lie between coding regions are called intervening sequences, or introns. The coding regions are called exons, because they are eventually expressed, usually by being translated into amino acid sequences. (1). Small nuclear ribonucleoproteins (snrnps) and other proteins form a molecular complex called a spliceosome on a pre-mrna molecule containing exons and introns. (2). Within the spliceosome, snrna base-pairs with nucleotides at specific sites along the intron. (3). The spliceosome cuts the pre-mrna, releasing the intron for rapid degradation, and at the same time splices the exons together. The spliceosome then comes apart, releasing mrna, which now contains only exons.

Regulation of genes in human: Alterative splicing.

Post translational modification Regulation of genes in human: Alterative splicing. Why alternative splicing important Post translational modification

UK(BBO)2015, 17D

1D. 2B. 3D. 4D.5E

The genetic code is the set of rules by which information encoded within mrna sequences is translated into proteins by living cells. Biological decoding is accomplished by the ribosome, which links amino acids in an order specified by mrna, using transfer RNA (trna) molecules to carry amino acids and to read mrna codon (three nucleotides) at a time. The genetic code is highly similar among all organisms and can be expressed in a table with 64 entries.

Translation: translation is the process in which cellular ribosomes create proteins. translation proceeds in four phases: Initiation: The ribosome assembles around the target mrna. The first trna is attached at the start codon(aug). Elongation: The trna transfers an amino acid to the trna corresponding to the next codon. Translocation: The ribosome then moves (translocates) to the next mrna codon to continue the process, creating an amino acid chain. Termination: When a stop codon is reached, the ribosome releases the polypeptide. Ribosome (fat man): I cannot move, because I have eaten too many amino acids mrna: protein translation law requires ribosomes to run from 5 to 3 direction on the mrna

Anticodon loop is mrna codon base-pairing site

The ribosome has three sites for trna to bind. They are the aminoacyl site ( A), the peptidyl site (P) and the exit site (E). With respect to the mrna, the three sites are oriented 5 to 3 E-P-A, because ribosomes move toward the 3' end of mrna.

An initiator trna serves to activate translation and occupies the P site. In all organisms the codon for the initiation of protein synthesis is AUG on mrna, which codes for the amino acid methionine. The P site holds the trna with the growing polypeptide chain. The A site binds the incoming trna with the complementary codon (UAC) of mrna. The E site holds the free trna without any amino acid.

Elongation: Then, a peptide bond forms between the amino acid of the trna in the A site and the amino acid of the charged trna in the P site. The growing polypeptide chain is transferred to the trna in the A site. Translocation occurs, moving the trna in the P site, now without an amino acid, to the E site; the trna that was in the A site, now charged with the polypeptide chain, is moved to the P site. The trna in the E site leaves and another aminoacyl-trna enters the A site to repeat the process.

Termination: The synthesis of a polypeptide is ended by stop codons on mrna. There are three stop codons on mrna (UAG, UAA, or UGA). The new polypeptide, free trna, mrna and ribosomal subunits are released. Ribosomes attached to the rough endoplasmic reticulum produce the proteins that are expelled either to the plasma membrane, or for organelles, or outside the cell altogether. The "free ribosomes" (not attached to rough ER) produce the proteins that stay inside the cell.

6A.7B. 8B. 9C. 10D.11A. 12C. 13C, 14 A

Ribosomes are composed of many special proteins and nucleic acids

UK(BBO)2015, 2B

Mutation: In biology, a mutation is a permanent alteration of the nucleotide sequence of the genome of an organism. There are various kinds of mutations. A point mutation, is an alteration in DNA sequence caused by a single nucleotide base change: base substitution, insertion, or deletion. A frameshift mutation is a genetic mutation caused by the insertion or deletion which can change the reading frame (the grouping of the codons), resulting in a completely different translation from the original. Point mutations can have one of three effects. 1. Silent mutation, the base substitution can be a silent mutation where the altered codon corresponds to the same amino acid. 2. Missense mutation, the base deletion or insertion can be a missense mutation where the altered codon corresponds to a different amino acid. 3. Nonsense mutation, the base deletion or insertion can be a nonsense mutation where the altered codon corresponds to a stop signal.

Chromosome Mutation: Aneuploidy and Polyploidy

14A

Chromosomal aberrations or Gene rearrangements 1. Deletions: A portion of the chromosome is missing or deleted. Known disorders in humans include Wolf-Hirschhorn syndrome, which is caused by partial deletion of the short arm of chromosome 4; and Jacobsen syndrome, also called the terminal 11q deletion disorder. 2. Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material. Known human disorders include Charcot-Marie-Tooth disease type 1A, which may be caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on chromosome 17. 3. Translocations: A portion of one chromosome is transferred to another chromosome. There are two main types of translocations: Reciprocal translocation: Segments from two different chromosomes have been exchanged. Robertsonian translocation: An entire chromosome has attached to another at the centromere - in humans these only occur with chromosomes 13, 14, 15, 21, and 22. 4. Inversions: A portion of the chromosome has broken off, turned upside down, and reattached, therefore the genetic material is inverted. 5. Transposons is a DNA sequence that can change its position within its genome.

Viruses A virus is a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. Bacteriophages or phages, are viruses that attach only bacteria. Structure of viruses 1. Capsid, a virus consists of nucleic acid surrounded by a protective coat of protein called a capsid. The capsid is made from proteins encoded by the viral genome. 2. A lipid "envelope, viruses can have a lipid "envelope" derived from the host cell membrane. 3. The viral genome, a virus has either a DNA or an RNA genome and is called a DNA virus or an RNA virus, respectively. They may be double stranded (dsdna or dsrna) or single-stranded (ssdna or ssrna).

Viral reproduction: The lytic cycle is one of the two cycles of viral reproduction, the other being the lysogenic cycle. The lytic cycle results in the destruction of the infected cell and its membrane. A key difference between the lytic and lysogenic cycles is that in the lytic phage, the viral DNA exists as a separate molecule within the bacterial cell, and replicates separately from the host bacterial DNA. The location of viral DNA in the lysogenic cycle is within the host DNA, therefore in both cases the virus/phage replicates using the host DNA machinery, but in the lytic phage cycle, the phage is a free floating separate molecule to the host DNA. HIV virus is ssrna retrovirus that can use both lytic and lysogenic cycles to replicate.

Transposons present in incoming DNA:

DNA repair in animal cells: three mechanisms are used to repair DNA replication errors 1. Proofreading by DNA polymerase. 2. Mismatch repair protein to errors that escape the proofreading. 3. Excision repair proteins identify and remove damaged nucleotides.

Prokaryotes: DNA replication is exemplified in E. coli. It is bidirectional and originates at a single origin of replication. Primers are needed to initiate the DNA replication.

Prokaryotes vs Eukaryotes :

Prokaryotes reproduction by binary fusion. Binary fission ("division in half") is a kind of asexual reproduction. It is the most common form of reproduction in prokaryotes and occurs in some single celled eukaryotes. After replicating its genetic material, the cell divides into two nearly equal sized daughter cells. The genetic material is also equally split. The daughter cells are genetically identical.

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.

Plasmid (1) Episome is a type of plasmid that can become incorporated in to the prokaryotic chromosome

Plasmid (2) 3. An antibiotic resistance gene (ampicillin) can be inserted into a plasmid.

Plasmid (3)

Restriction enzyme

Restriction enzyme EcoRI recognizes the sequence

A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. They are most commonly found in bacteria as small, circular, double-stranded DNA molecules; however, plasmids are sometimes present in archaea and eukaryotic organisms. R plasmids, provide bacteria with resistance against antibiotics.

Biotechnology: Gel electrophoresis

Biotechnology: The polymerase chain reaction (PCR) 1 3 2

Biotechnology: The polymerase chain reaction (PCR) Taq polymerase, a DNA polymerase derived from thermophilic bacteria, is used in polymerase chain reaction (PCR) in the laboratory. During PCR, Taq polymerase catalyzes DNA polymerization, similar to how it would in bacteria. A normal PCR cycle is as follows: Melting/denaturing 95 C Primer annealing 50 C Elongation 72 C Repeat 30 cycles Which of the following conditions likely describes the living environment of Taq bacteria? a. Freshwater with acidic ph b. Hydrothermal vents reaching temperatures between 70-75 C c. Hot springs of 40 C d. Tide pools with high salinity

Biotechnology: DNA cloning Cloning DNA of interest into a kind of plasmids Transformation with a plasmid containing DNA of interest Restriction enzymes are enzymes that cut a DNA molecule at a particular place. Such as HamH1 and Hae III below:

Biotechnology:

Biotechnology: RFLP In molecular biology, Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases.

Biotechnology: DNA fingerprinting is a laboratory technique used to establish a link between biological evidence and a suspect in a criminal investigation. A DNA sample taken from a crime scene is compared with a DNA sample from a suspect. If the two DNA profiles are a match, then the evidence came from that suspect.

Biotechnology: DNA Fingerprinting

Biotechnology: Human genome project The Human Genome Project (HGP) is an international scientific research project with the goal of determining the sequence of chemical base pairs which make up human DNA, and of identifying and mapping all of the genes of the human genome from both a physical and functional standpoint.

Biotechnology: Beyond the Human genome project The sequencing of the human genome holds benefits for many fields, from molecular medicine to human evolution. The Human Genome Project, through its sequencing of the DNA, can help us understand diseases including: genotyping of specific viruses to direct appropriate treatment; identification of mutations linked to different forms of cancer; the design of medication and more accurate prediction of their effects

15B.16A. 17D. 18D. 19C.20A.

1. A yeast cell, which encodes a DNA polymerase that is able to add nucleotides in both the 5 to 3 and 3 to 5 direction. Which of the following structures would this cell not like to generate during DNA replication? a. RNA primers b. Okazaki fragments c. Replication d. Nicked DNA by topoisomerases 2. A eukaryotic gene, which does not normally undergo splicing, was exposed to benzyrene, a known carcinogen and mutagen. Following exposure, the protein encoded by gene was shorter than before exposure. Which of the following types of genetic rearrangements or mutations was likely introduced by the mutagen? a. Silent mutation b. Missense mutation c. Nonsense mutation d. Duplication 3. Which of the following represents the correct order beginning with the earliest activity of enzymes involved in DNA replication. a. Helicase, ligase, RNA primase, DNA polymerase b. DNA polymerase, RNA primase, helicase, ligase c. RNA primase, DNA polymerase, helicase, ligase d. Helicase, RNA primase DNA polymerase, ligase 4. If a messenger RNA codon is UAC, which of the following would be the complementary anticodon triplet in the trna? a. ATG b. AUC c. AUG d. ATT

5.During post-translational modification, the polypeptides from a eukaryotic cell typically undergoes substantial alteration that results in a. Excision of introns b. Addition of poly(a) tail c. Formation of peptide bonds d. A change in the overall conformation of a polypeptide 6. Which of the following represents the maximum number of amino acids that could be incorporated into polypeptide encoded by 21 nucleotides of mrna? a. 3 b. 7 c. 21 d. 42 7. A research uses molecular biology techniques to insert a human lysosomal membrane protein into bacterial cells to produce large quantities of this protein for later study. However, only small quantities of this protein result in these cells. What is a possible explanation for this result? a. The membrane protein requires processing in the ER and Golgi. Which are meaning in the bacterial cells. b. Bacteria do not make membrane proteins c. Bacteria do not use different transcription factors than human, so the gene was not expressionbacteria do not have enough trnas to make this protein sequence. 8. Viruses and bacteria have which of the following in common?? a. Ribosomes b. Nucleic acid c. Flagella d. Metabolism

5. During post-translational modification, the polypeptides from a eukaryotic cell typically undergoes substantial alteration that results in a. Excision of introns b. Addition of poly(a) tail c. Formation of peptide bonds d. A change in the overall conformation of a polypeptide 6. Which of the following represents the maximum number of amino acids that could be incorporated into polypeptide encoded by 21 nucleotides of mrna? a. 3 b. 7 c. 21 d. 42 7. A research uses molecular biology techniques to insert a human lysosomal membrane protein into bacterial cells to produce large quantities of this protein for later study. However, only small quantities of this protein result in these cells. What is a possible explanation for this result? a. The membrane protein requires processing in the ER and Golgi. Which are meaning in the bacterial cells. b. Bacteria do not make membrane proteins c. Bacteria do not use different transcription factors than human, so the gene was not expressionbacteria do not have enough trna s to make this protein sequence. 8. Viruses and bacteria have which of the following in common?? a. Ribosomes b. Nucleic acid c. Flagella d. Metabolism

Transcriptional regulation: Transcription is an essential step in gene expression and its understanding has been one of the major interests in molecular and cellular biology. By precisely tuning gene expression, transcriptional regulation determines the molecular machinery for developmental plasticity, homeostasis and adaptation. Regulation of gene expression in prokaryotes 1. Promoter: The DNA region to which the RNA polymerase attaches to start transcription 2. Operator: The DNA region to which a regulatory protein (repressor or activator) attaches to block or promote the action of RNA polymerase 3. Structure gene: contains coding DNA for mrna 4. Regulator gene: Lying outside the operon region, produces a regulatory protein. The Repressor blocks the attachment of RNA polymerase to the promoter and inhibits the transcription. The Activator promotes the attachment of RNA polymerase to the promoter and enhances the transcription.

Trp (trptophan) operon are an example of negative regulation. An example of a regulatory sequence is the TATA box. an example of a regulatory protein is the TATA binding protein. Trp operon has 5 genes. When trptophan (trp) present it binds to the repressor which then binds to the operator preventing synthesis of more trp. When trp absent repressor changes shape and does not bind to the operator, which allows RNA polymerase to bind to the promotor to make RNA and then proteins. the genes in the trp operon are only expressed in the absence of tryptophan. A mutation disrupts a codon in the trp regulatory gene: no repressor is produced, genes in the trp operon are always expressed. Mutation disrupts operator sequence of the trp operator but RNA polymerase can still bind repressor cannot bind so genes are always expressed Q1. The Lac operon in E.coli is involved in a. Regulating the expression of a gene. b. Regulating the translation of mrna. c. Controlling the formation of ribosomes. d. Controlling DNA replication

The lac operon is also an examples of negative regulation An example of a regulatory sequence is the TATA box.an example of a regulatory protein is the TATA binding protein the lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli. lac operon has 3 genes lacz, lacy and laca; lacz encodes β- galactosidase, lacy encodes lactose permease, laca encodes galactoside O-acetyltransferase. when lactose is present allolactose binds to the repressor, changing its form and causes it to drop from the operator. RNA polymerase then binds to the promotor and makes RNA which makes proteins. When no lactose present, repressor bound to operator. Mutation disrupts a codon in the laci gene(produces repressor) repressor cannot bind so genes are always expressed A base pair insertion occurs in the promotor of the lac operon, but it may not affect the binding of RNA polymerase or the repressor. Because no effect on the coding sequence A base pair insertion in the lacz coding sequence causes frameshift that affects the lacz protein only because each gene has their own start and stop codon

When there is lactose present but no glucose laci repressor does not bind but CAP does and there is transcription of the lac operon. Cap protein is an activator. Cap and camp are samples of positive gene regulation When there is lactose and glucose present neither the laci repressor nor the CAP protein bind, there is no transcription of the lac operon in the lac operon: low glucose, high camp. camp binds to CAP, CAP activated.

Q2-5. refer to operon a. Lactose. b. Repressor. c. Regulatory gene. d. Promoter. Q2. Acts as an inducer in lac operon. Q3. Binding site for RNA polymerase. Q4. Codes for the repressor. Q5. Binds at the operator.

Q2-5. refer to operon a. Lactose. b. Repressor. c. Regulatory gene. d. Promoter. Q2. Acts as an inducer in lac operon. A Q3. Binding site for RNA polymerase. D Q4. Codes for the repressor. C Q5. Binds at the operator.b

Regulation of genes in human is far more complex.

Regulation of genes in human: Chromatin modification-acetylation of histone tails.

Regulation of gene expression DNA methylation

Regulation of genes in human: Chromatin modification-dna methylation.

The non-coding RNAs (ncrna) are RNAs that does not encode a protein, but this does not mean that such RNAs do not contain information nor have function. Although it has been generally assumed that most genetic information is transacted by proteins, recent evidence suggests that the majority of the genomes of mammals and other complex organisms is in fact transcribed into ncrnas, many of which are alternatively spliced and/or processed into smaller products. These ncrnas include micrornas and snornas (many if not most of which remain to be identified), as well as likely other classes of yet-to-be-discovered small regulatory RNAs, and tens of thousands of longer transcripts (including complex patterns of interlacing and overlapping sense and antisense transcripts), most of whose functions are unknown. These RNAs (including those derived from introns) appear to comprise a hidden layer of internal signals that control various levels of gene expression in physiology and development, including chromatin architecture/epigenetic memory, transcription, RNA splicing, editing, translation and turnover. RNA regulatory networks may determine most of our complex characteristics, play a significant role in disease and constitute an unexplored world of genetic variation both within and between species. The non-coding RNAs (ncrna)