Nucleic Acids and Proteins PDF

NUCLEIC ACIDS AND PROTEINS Primary Structure of Nucleic Acid Nucleic acids - are polymers of nucleotides (nucleosides with a phosphate group bonded to -OH on C5') - have a unique sequence of bases in RNA, which is called its primary structure - carry information from one cel...

NUCLEIC ACIDS AND PROTEINS Primary Structure of Nucleic Acid Nucleic acids - are polymers of nucleotides (nucleosides with a phosphate group bonded to -OH on C5') - have a unique sequence of bases in RNA, which is called its primary structure - carry information from one cell to the next Primary Structure of Nucleic Acids In the primary structure of nucleic acids, - nucleotides are joined by a 3'-5' phosphodiester bond - the 3'-OH group of the sugar in one nucleotide forms an ester bond to the phosphate group on the 5'-carbon of the sugar of the next nucleotide Example of Phosphodiester Bonds - The following nucleotide sequence starting with adenine (free -5'-phosphate end) is 5'-A-C-G-U-3', joined by phosphodiester bonds. In the primary structure of nucleic acids in DNA and RNA chains, - each sugar in a sugar-phosphate backbone is attached to a base - the bases extend out from the nucleic acid backbone - are labeled starting with the free 5' end to the 3' end.  In this nucleic acid, the sequence of bases is represented by 5'-A-C-G-U-3'.In the primary structure of RNA, A, C, G, and U are linked by 3-5'-phosphodiester bonds. In a nucleic acid the nucleotides are joined by a phosphodiester bond that joins the 3'-OH group of the sugar to the phosphate group on the 5C' on the next nucleotide. Double Helix of DNA & DNA Replication DNA Double Helix DNA contains complementary base pairs, equal amounts of A and T and equal amounts of G and C bases in which - adenine is always linked by two hydrogen bonds with thymine (A-T) - guanine is always linked by three hydrogen bonds with cytosine (G-C)  A double helix - has two strands of nucleotides that wind together - is held in place by two hydrogen bonds that form between the base pairs A-T - is held in place by three hydrogen bonds that form between the base pairs G-C In the double helix of DNA, - the two chains are held together by hydrogen bonds that link bases A-T and G-C - the bases along one strand complement the bases along the other DNA Replication In DNA replication, - genetic information is maintained each time a cell divides - the DNA strands unwind - each parent strand bonds with new complementary bases - two new DNA strands form that are exact copies of the parent DNA In DNA replication, the separate strands of the parent DNA are the templates for the synthesis of complementary strands, which produces two exact copies (daughter DNAs). DNA Fingerprinting In a process called DNA fingerprinting, - enzymes are used to cut DNA chains into smaller sections - the resulting fragments are separated by size and treated with a radioactive isotope that adheres to specific base sequences - the resulting pattern of bands is known as a DNA fingerprint One application of DNA fingerprinting is in forensic science, where DNA from samples such as blood, hair, or semen is used to connect a suspect with a crime. Dark and light bands on X-ray film show up as a result of the radioactive isotope that adheres to specific sequences. RNA Types Ribonucleic acid (RNA) - is a nucleic acid similar to DNA, but with only a single, helical strand of bases. It plays a key role in turning DNA instructions into functional proteins. Structures of RNA Single Stranded Structure Made up of Nucleotides: -Ribose -Phosphate Group -Nitrogenous Base (Adenine, Uracil, Guanine, Cytosine) TYPES OF RNA 1) Messenger RNA (mRNA) - Messenger RNA (mRNA) molecules are produced in the process called transcription, and they carry the genetic information from the DNA in the nucleus directly to the cytoplasm, where most of the protein is synthesized. 2) Transfer RNA (tRNA) - is a small RNA molecule that plays a key role in protein synthesis. - Transfer RNA serves as a link between the messenger RNA molecule and the growing chain of amino acids that make up a protein. 3) Ribosomal RNA (rRNA) - is part of the ribosome, or protein builders of the cell. - rRNA are responsible for reading the order of amino acids and linking amino acids together. PROTEIN SYNTHESIS: Transcription - is the process of copying a segment of DNA into mRNA - First step in gene expression that leads to the creation of proteins EUKARYOTIC CELL PROKARYOTIC CELL Synthesis of mRNA 1. Pre-mRNA PRE-MESSENGER RNA INITIATION - RNA polymerase ---- Binds to promoter ELONGATION - Copying the basis on the DNA template strand to form new complementary RNA strand TERMINATION - Termination Region signals when to stop PROCESS NUCLEOTIDES  2. MATURATION OF PRE- MRNA Regulation of Transcription Why Regulate Transcription? - To ensure genes are expressed at the right time, in the right cells, and at the appropriate levels. MAJOR PLAYERS DNA TEMPLATE information - Provides the genetic information RNA Polymerase - Enzyme that synthesizes RNA from the DNA template PROMOTER REGION - DNA sequence where RNA polymerase binds to initiate transcription TRANSCRIPTION FACTORS - Proteins that help regulate the initiation of transcription. PROMOTERS AND ENHANCERS Promoters: - Located near the gene start site, where RNA polymerase binds. Enhancers and Silencers: - Enhancers - increase transcription. - Silencers - decrease transcription. TRANSCRIPTION FACTORS ACTIVATORS: Bind to enhancer regions to increase transcription. REPRESSORS: Bind to silencer regions to decrease transcription. GENERAL TRANSCRIPTION FACTORS (GTFS): Required for basic transcription machinery (e.g., tfiid in eukaryotes). SPECIFIC TRANSCRIPTION FACTORS: Activated by signaling pathways (e.g., hormones or stress responses) REMODELLING OF CHROMATIN - DNA is wrapped around histones to form chromatin splicing SPLICING - Increases protein diversity from a single gene. - Regulated to produce different protein isoforms in different cell types or conditions. mRNA Processing (Exons and Introns) mRNA Processing refers to a series of modifications that occur to messenger RNA (mRNA) molecules after they are transcribed from DNA but before they are translated into proteins. This process is critical for the maturation and stability of the mRNA, as well as for its eventual translation > Pre-mRNAs are first coated in RNA-stabiliz-ing proteins > The three most important steps of pre-mRNA processing are the addition of stabilizing and signaling factors at the 5' and 3' ends of the mole-cule, and the removal of the introns. 5’ Capping While the pre-mRNA is still being synthesized, a 7-methylguanosine cap, also called the 5' cap, is added to the 5′ end of the growing transcript by a phosphate linkage. This functional group protects the nascent mRNA from degradation. In addition, factors involved in protein synthesis recognize the cap to help initiate translation by ribosome 3’ Poly-A-Tail Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the pre-mRNA. An enzyme called poly-A polymerase then adds a string of approximately 200 A residues, called the poly-A tail This modification further protects the pre-mRNA from degradation and is also the binding site for a protein necessary for exporting the processed mRNA to the cytoplasm. Splicing Pre-mRNA often contains non-coding sequences called introns, which need to be removed. Splicing is the process by which introns are excised, and the remaining coding sequences, called exons, are joined together to form a continuous coding sequence. This process is carried out by the spliceosome, a complex of small nuclear RNA (snRNA) and proteins LACTOSE OPERON The lac operon codes for enzymes involved in the catabolism The presence of lactose induces the operon to switch on Inducible operons have proteins that can bind to either activate or repress transcription depending on the local environment and the needs of the cell. Designated Genes (Enzyme) of the Lac Operon (3 enzymes required to metabolize lactose) 1. lac z: B-galactosidase - Breaks down lactose to - glucose and galactose 2. lac y: galactose permease - Found in the E.coli cytoplasmic membrane and actively transports lactose into the cells 3. lac a: thio-galactoside transacetylase - The function of this enzyme is unknown but it is coded for by the gen lacA. Elements of the Operon Operator (lacO): Binding sites for repressor Promoter (lacP): Binding site for RNA polymerase Repressor: Binds to DNA at the operator & blocks binding of RNA Polymerase at the promoter. lacI: Controls production of repressor protein Function of Lac Operon In the absence of lactose (inducer), the regulator gene produces a repressor protein which binds to the operator site & prevents the transcription as a result, the structural gene does not produce mRNA & the proteins are not formed. When lactose (inducer), introduced in the medium, binds to the repressor the repressor now fails to bind to the operator. Therefore the operator is made free & induces the RNA polymerase to bind to the initiation site on the promoter which results in the synthesis of lac mRNA. This mRNA codes for three enzymes necessary for lactose catabolism. 1. When lactose is absent - A repressor protein is continuously synthesized. It sits on a sequence of DNA just in front of the lac operon, the Operator site - The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing 2. When lactose is present - A small amount of sugar allolactose is formed within the bacterial cell, this fits onto the repressor protein at another active site (allosteric site) - This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site 3..When both glucose and lactose are present - When glucose and lactose are present RNA polymerase can sit on the promoter site but it is unstable and it keeps falling off. 4. When glucose is absent and lactose is present - Another protein is needed, an activator protein which stabilizes RNA polymerase. - The activator protein only works when glucose is absent and in this way E. coli only makes enzymes to metabolize other sugars in the absence of glucose Positive Control - This binding is essential for the transcription of the structural gene. - lacP- mutations are cis acting. Negative Control - The lac repressor binds to the operator. - The DNA sequence covered by the repressor overlaps the DNA sequence recognized by the RNA polymerase. Lac Mutations Some part of the lac operon are cis acting where others are trans acting. Structural-Gene Mutation The mutation which occurred on lacz and lacy structural genes altered the amino acid sequences of the proteins encoded by the genes. a. In the absence of inducer, the laco+ operon is turned off, whereas the lacoc operon produces functional ẞ-galactosidase from the lacZ+ gene and nonfunctional permease molecules from the lacy gene with missense mutation. b. In the presence of inducer the functional B- galactosidase and defective permease are produce from the laco operon, whereas the laco+ operon produces nonfunctional ẞ-galactosidase from the lacz gene & functional permease from lacY+ gene Translation (tRNA activition and translocation) and Termination Translation The process by which the genetic information preserved in the dna and transcribed into mrna is converted to the language of proteins. Genetic Code The Genetic Code is read In triplets of nucleotides, known as codons.Each codon corresponds to a specific amino acid or serves as a signal to start or stop protein synthesis. For Example,the codon AUGcodesforthe amino acid methionine and also signals the start of protein synthesis 2 Major Processes of Translation 1. Trna activation (aminoacylation) - The process by which a trna molecule charged with its corresponding amino acid. - trna transfers the amino acids to the ribosomes so it can have a polypeptide chain 2. Translocation - The whole ribosome moves one codon along the mRNA. Simultaneously with this move, the dipeptide is translocated from the A site to the P site. - The empty tRNA is moved to the E site. - When this cycle occurs one more time, the empty tRNA will be ejected and go back to the pool of tRNA that is available for activation with an amino acid. Termination - The polypeptide continues to grow by way of translocation until all necessary amino acids are in place and bonded to each other. - Appearance in the mRNA codon sequence of one of the three stop codons (UAA, UAG, or UGA) terminates the process. - No tRNA has an anticodon that can base-pair with these stop codons. - The polypeptide is then cleaved from the tRNA through hydrolysis Gene Mutation and Diseases Mutation Occur when mistakes happen during the DNA copying process Alters the nucleotide sequence in DNA.  Results from mutagens such as radiation and chemicals. Produces one or more incorrect codons (sets of three nucleotides that form a code, guiding cells in protein production) in the corresponding mRNA, producing a protein that incorporates one or more incorrect amino acids. Causes genetic diseases that produce defective proteins and enzymes. Normal DNA Sequence Mutation: Substitution A different base substitutes for the proper base in DNA. There is a change in a codon in the mRNA. The wrong amino acid may be placed in the polypeptide. Mutation: Frameshift An extra base adds to or is deleted from the normal DNA sequence. All the codons in mRNA and amino acids are incorrect from the base change. Genetic Diseases: 1. Huntington’s Disease - Appearing in middle age, HD affects the nervous system, leading to total physical impairment. It is the result of a mutation in a gene on chromosome 4, which can now be mapped to test people in families with HD. 2. Cystic Fibrosis - The most common inherited disease. Thick mucus secretions make breathing difficult and block pancreatic function.  3. Down Syndrome - The leading cause of mental retardation, occurring in about 1 of every 800 live births. Mental and physical problems including heart and eye defects are the result of the formation of three chromosomes, usually chromosome 21, instead of a pair. 4. Hemophilia - One or more defective blood-clotting factors lead to poor coagulation, excessive bleeding, and internal hemorrhages. Recombinant DNA and its Products Recombinant DNA In recombinant DNA, A DNA fragment from one organism is combined with DNA in another. Restriction enzymes are used to cleave a gene from a foreign DNA and open DNA plasmids in E. coli. DNA fragments are mixed with the plasmids in E. coli and the ends are joined by ligase. The new gene in the altered DNA produces protein. Product of Recombinant DNA DNA Fingerprinting and polymerase chain reaction What is DNA Fingerprinting? DNA Fingerprinting is a forensic technique used to identify individuals by characteristics of their DNA. Also called DNA Profiling or Molecular Fingerprinting. DNA Fingerprinting patterns DNA Profile V victim S sample from crime scene S1 suspect 1 S2 suspect 2 S3 suspect 3 Paternity test 1 mother 2 son 3 possible father A 4 possible father B There is a match between one of the child’s restriction Fragments and one of the mother ’s. Polymerase Chain Reaction What is PCR? PCR is a technique that takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing. In vitro technique Condition 1. Denaturation of ds DNA template 2. Annealing of primers 3. Extension of ds DNA molecules Denaturation Temperature: 92-94C Double stranded DNA melts → single stranded DNA Annealing Temperature: ~50-70C (dependent on the melting temperature of the expected duplex) Primers bind to their complementary sequences. Extension Temperature: ~72C Time: 0-5-3min DNA polymerase binds to the annealed primers and extends DNA at the 3’ end of the chain. Products of Extension Overall Principle of PCR DNA - 1 copy ○ Known sequence Sequence of interest known sequence PCR Virus and Reverse Transcription Viruses are small particles of DNA or RNA that require a host cell to replicate cause a viral infection when the DNA or RNA enters a host cell are synthesized in the host cell from the viral RNA produced by viral DNA. Some Diseases Caused by Viruses Some Diseases Caused by Viral Infection Disease Virus Common Cold Coronavirus (over 100 types Influenza Orthomyxovirus Warts Papovavirus Herpes Herpesvirus HPV Human papillomavirus Leukemia, cancers, AIDS Retrovirus Hepatitis Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) Mumps Paramyxovirus Epstein-Barr Epstein-Barr virus (EBV) Chicken pox (shingles) Varicella zoster virus (VZV) Viruses After a virus attaches to the host cell, it injects its viral DNA and uses the host cell's amino acids to synthesize viral protein. It uses the host cell's nucleic acids, enzymes, and ribosomes to make viral RNA. When the cell bursts, the new viruses are released to infect other cells. Reverse Transcription In reverse transcription, a retrovirus, which contains viral RNA but no viral DNA, enters a cell the viral RNA uses reverse transcriptase to produce a viral DNA strand the viral DNA strand forms a complementary DNA strand the new DNA uses the nucleotides and enzymes in the host cell to synthesize new virus particles Reverse Transcription After a retrovirus injects its viral RNA into a cell, it forms a DNA strand by reverse transcription. The DNA forms a double-stranded DNA called a provirus, which joins the host cell DNA. When the cell replicates, the provirus produces the viral RNA needed to produce more virus particles. HIV Virus and AIDS The HIV-1 virus is a retrovirus that infects T4 lymphocyte cells decreases the T4 level and the immune system fails to destroy harmful organisms causes pneumonia and skin cancer associated with AIDS AIDS Treatment Treatment for AIDS is based on attacking HIV-1 at different points in its life cycle, such as prevention of reverse transcription of the viral DNA and protein synthesis. For example, AZT, similar to thymidine, mimics the structures of the nucleosides used for DNA synthesis, which inhibit the reverse transcriptase enzyme. Lexiva is a protease inhibitor that prevents protein synthesis used by viruses to make more copies. Concept Map: Nucleic Acids and Protein Synthesis Conclusion Viruses are small agents that require host cells to replicate, injecting their genetic material to produce new viral particles. Retroviruses like HIV convert their RNA into DNA through reverse transcription, integrating into the host genome and impairing the immune system, leading to AIDS. Treatments target HIV's life cycle stages, using drugs like AZT and Lexiva to inhibit replication. Understanding these processes is essential for effective antiviral strategies. HIV Virus and AIDS Treatment What is HIV? HIV stands for Human Immunodeficiency Virus, a virus that attacks the body’s immune system, specifically the CD4 cells (T-cells), which help the body fight off infections. What does HIV do to a person? HIV attaches to CD4 T cells and uses their machinery to replicate. This replication leads to the death of infected T cells.