Nucleic Acids PDF

Nucleic Acids Structure of the Nucleotide DNA and RNA are polymers whose monomer units are called nucleotides A nucleotide itself consists of: 1. a nitrogen containing heterocyclic base 2. a ribose or deoxyribose sugar ring 3. a phosphoric acid unit DNA and RNA Deoxyribonucleic acid (DNA)...

Nucleic Acids Structure of the Nucleotide DNA and RNA are polymers whose monomer units are called nucleotides A nucleotide itself consists of: 1. a nitrogen containing heterocyclic base 2. a ribose or deoxyribose sugar ring 3. a phosphoric acid unit DNA and RNA Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the chemical carriers of genetic information Nucleic acids are biopolymers made of nucleotides, aldopentoses linked to a purine or pyrimidine and a phosphate Sugars in DNA and RNA RNA is derived from ribose DNA is from 2-deoxyribose (the ' is used to refer to positions on the sugar portion of a nucleotide) Major Purine Bases Major Pyrimidine Bases Nucleotides A nucloetide is the repeating unit of the DNA or RNA polymer. In DNA and RNA the base is bonded to C1 of the sugar and the phosphate is bonded to C5 (and connected to 3’ of the next unit) Naming Nucleotides Begin with the name of the nitrogenous base. Remove –ine ending and replace with: –-osine for purines or –idine for pyramidines. Uracil: -acil with –idine. ribose then ribonucleotide –deoxyribose then deoxyribonucleotide –deoxy before base name for deoxyribonucleotide Add prefix for number of phosphoryl groups –Monophosphate, diphosphate, triphosphate The Deoxyribonucleotides The Ribonucleotides Generalized Structure of DNA DNA-Secondary Structure The most common form of DNA is the  form. Its structure was determined by Watson and Crick in 1953. This DNA consists of two chains of nucleotides coiled around one another in a right handed double helix. The chains run antiparallel and are held together by hydrogen bonding between complementary base pairs: A=T, G=C. H-bonding in DNA Structure Hydrogen bonding between A and T or G and C helps to hold the chains in the double helix The strands are said to be complementary H-Bonds in DNA The G-C base pair involves three H-bonds A-T Base Pairing Involves two H-bonds  B DNA segment Sugar-phosphate Chain 2 backbone Chain 1 Hydrogen bonded base pairs in the core of the helix Grooves in DNA The strands of the DNA double helix create two continuous grooves (major and minor) The sugar–phosphate backbone runs along the outside of the helix, and the amine bases hydrogen bond to one another on the inside The major groove is slightly deeper than the minor groove, and both are lined by potential hydrogen bond donors and acceptors.  B DNA Structure Major groove Outside diameter, 2 nm Interior diameter, 1.1 nm Minor groove Length of one turn of helix is 3.4 nm and contains 10 base pairs. Nucleic Acid Sequences Differences arise from the sequence of bases on the individual nucleotides Describing a Sequence Chain is described from 5 end, identifying the bases in order of occurrence, using the abbreviations A for adenosine, G for guanosine, C for cytidine, and T for thymine (or U for uracil in RNA) A typical sequence is written as TAGGCT  Learning Check NA1 Write the complementary base sequence for the matching strand in the following DNA section: -A-G-T-C-C-A-A-T-G-C- 22  Solution NA1 Write the complementary base sequence for the matching strand in the following DNA section: -A-G-T-C-C-A-A-T-G-C- -T-C-A-G-G-T-T-A-C-G- 23 Chromosomes Chromosomes are pieces of DNA that contain the genetic instructions, or genes, of an organism. Prokaryotes (single chromosome) – No true nucleus. Chromosome is a circular DNA molecule that is supercoiled, that is, the helix is coiled on itself. – At approximately 40 sites a complex of proteins is attached, forming a series of loops. – This structure is the nucleoid. Chromosomes Eukaryotes (Number and size of chromosomes vary.) – True nucleus. Membrane bound organelles that separate cellular functions. – Nucleosome which consists of a strand of DNA wrapped around a disk of histone proteins. – Larger structure is the 30 nm fiber. – Coiled in to a 200 nm fiber RNA Structure Sugar-phosphate backbone for ribonucleotides linked by 3’-5’ phosphodiester bonds. – RNA molelcules usually single stranded. – Ribose replaces deoxyribose. – Uracil replaces thymine. Base pairing between U and A and G and C results in portions of the single strand that become double stranded. Nucleic Acids and Heredity Processes in the transfer of genetic information: Replication: identical copies of DNA are made Transcription: genetic messages are read and carried out of the cell nucleus to the ribosomes, where protein synthesis occurs. Translation: genetic messages are decoded to make proteins.  DNA Replication DNA in the chromosomes replicates itself every cell division Maintains correct genetic information Two strands of DNA unwind Each strand acts like a template New bases pair with their complementary base Two double helixes form that are copies of original DNA 28 OVERVIEW: DNA Unwinds G-C G- -C A-T A- -T C-G C- -G T-A T- -A 29 DNA Copied with Base Pairs Two copies of the original DNA strand G-C G-C A-T A-T C-G C-G T-A G-A 30  Summary: DNA Replication 1. Opening up the superstructure. – During replication, the very condensed superstructure of chromosomes is opened by a signal transduction mechanism. – One step of this mechanism involves acetylation and deacetylation of key lysine residues. + acetylatio n Lys- CH2 CH2 CH2 CH2 NH3 Ly sin e side ch ain deacety lation (has a pos itive charge) O Lys-CH2 CH2 CH2 CH2 NH- CCH 3 Acety lated ly sine s ide chain (has no charg e) – Acetylation removes a positive charge and thus weakens the DNA-histone interactions. Summary: DNA Replication 2. Relaxation of higher structures of DNA. – Tropoisomerases (also called gyrases) facilitate the relaxation of supercoiled DNA by introducing either single strand or double strand breaks in the DNA. – Once the supercoiling is relaxed by this break, the broken ends are joined and the tropoisomerase diffuses from the location of the replication fork. Summary: DNA Replication 3. Unwinding the DNA double helix. – Replication of DNA starts with unwinding of the double helix. – Unwinding can occur at either end or in the middle. – Unwinding proteins called helicases attach themselves to one DNA strand and cause separation of the double helix. – The helicases catalyze the hydrolysis of ATP as the DNA strand moves through; the energy of hydrolysis promotes the movement. Summary: DNA Replication Primer/primases – Primers are short oligonucleotides— 4 to 15 nucleotides long. – They are required to start the synthesis of both daughter strands. – Primases are enzymes that catalyze the synthesis of primers. – Primases are placed at about every 50 nucleotides in the lagging strand synthesis. Summary: DNA Replication DNA polymerases are key enzymes in replication. – Once the two strands have separated at the replication fork, the nucleotides must be lined up in proper order for DNA synthesis. – In the absence of DNA polymerase, alignment is slow. – DNA polymerase provides the speed and specificity of alignment. – Along the lagging (3’ -> 5’) strand, the polymerases can synthesize only short fragments, because these enzymes only work from 5’ -> 3’. – These short fragments are called Okazaki fragments. – Joining the Okazaki fragments and any remaining nicks is catalyzed by DNA ligase. Mutation and Repair Mutations are mistakes introduced into the DNA sequence of an organism. Heritable change in the sequence of DNA. Can occur during replication They can be classified as: – Point: substitution of a single nucleotide for another. – Deletion: one or more nucleotides are lost. – Insertion: one or more nucleotides are added. Many mutagens (chemicals causing a change in the DNA sequence) are also carcinogens and cause cancer. Mutagens are chemicals that can cause a base change in DNA. UV Damage and DNA Repair UV light causes formation of a pyrimidine dimer on a DNA strand. Failure to repair this defect can lead to xeroderma pigmentosum. People who suffer from this genetic skin disorder are very sensitive to UV light and Note: Not all mutations are harmful. Certain ones may be beneficial because develop multiple they enhance the survival rate of the skin cancers. species. DNA Repair The viability of cells depends on DNA repair enzymes that can detect, recognize, and repair mutations in DNA. Base excision repair (BER): one of the most common repair mechanisms. – A specific DNA glycosylase recognizes the damaged base. – It catalyzes the hydrolysis of the -N-glycosidic bond between the incorrect base and its deoxyribose. – It then flips the damaged base, completing the excision – The sugar-phosphate backbone remains intact. DNA Repair BER (cont’d) – At the AP (apurinic or apyrimidinic) site thus created, an endonuclease catalyzes the hydrolysis of the backbone – An exonuclease liberates the sugar-phosphate unit of the damaged site – DNA polymerase inserts the correct nucleotide – DNA ligase seals the backbone to complete the repair NER (nucleotide excision repair) removes and repairs up to 24-32 units by a similar mechanism involving a number of repair enzymes The Central Dogma Classes of RNA Structure transfer RNA (tRNA) – Transfers amino acids to the site of protein synthesis (ribosomes). Has the anticodon. ribosomal RNA (rRNA) – rRNA forms ribosomes by reacting with proteins messenger RNA (mRNA) – mRNA directs the AA sequence of proteins and is a complimentary copy of a gene. It has the codon for an AA in a protein. tRNA There is at least one tRNA (and often several) for each AA to be incorporated into a protein. tRNA is single stranded with typically about 80 nucleotides. Intrachain hydrogen bonding (A=U and G=C) occurs to gives regions called stems with an -helix The overall structure is called a cloverleaf in a L-shaped conformation. tRNA Transfer RNA (tRNA) transfers AA to the site of protein synthesis. Has the anticodon Attachment to mRNA here AA attaches here Summary: Transcription Transcription: the process by which information encoded in a DNA molecule is copied into an mRNA molecule. – Transcription starts when the DNA double helix begins to unwind near the gene to be transcribed. – Only one strand of the DNA is transcribed. – Ribonucleotides assemble along the unwound DNA strand in a complementary sequence. – Enzymes called polymerases (poly) catalyze transcription: poly I for rRNA formation, poly II for mRNA formation, and poly III for tRNA formation. Summary: Transcription A eukaryotic gene has two parts: – A structural gene that is transcribed into RNA; the structural gene is made of exons and introns. – A regulatory gene that controls transcription; the regulatory gene is not transcribed but has control elements, one of which is the promoter. – A promoter is unique to each gene. – There is always a sequence of bases on the DNA strand called an initiation signal. – Promoters also contain consensus sequences, such as the TATA box, in which the two nucleotides T and A are repeated many times. Summary: Transcription – A TATA box lies approximately 25 base pairs upstream (Figure 25.2). – All three RNA polymerases interact with their promoter regions via transcription factors that are binding proteins. – After initiation, RNA polymerase zips up the complementary bases in a process called elongation. – Elongation is in the 5’ -> 3’ direction. – At the end of each gene is a termination sequence. Summary: Transcription Poly II has two different forms: – At its C-terminal domain, it has Ser and Thr repeats that can be phosphorylated. – When poly II starts initiation, it is unphosphorylated. – Upon phosphorylation, it catalyzes elongation. – After termination of the transcription, it is dephosphorylated by a phosphatase. – In this manner, poly II is constantly recycled between its initiation and elongation roles. Summary: Transcription The RNA products of transcription are not necessarily functional RNAs. – They are made functional by post-transcription modification. – Transcribed mRNA is capped at both ends. – The 5’ end acquires a methylated guanine. – The 3’ end acquires a polyA tail that may contain from 100 to 200 adenine residues. – Once the two ends are capped, the introns are spliced out. – tRNA is similarly trimmed, capped, and methylated. – Functional rRNA also undergoes post-transcription methylation. The Genetic Code (DNA) The message on DNA translated to mRNA: 1. Degenerate: more than one three base codon can code for the same AA. 2. Specific: each codon specifies one AA 3. Nonoverlapping and commaless : none of the bases are shared between consecutive codons and no noncoding bases appear in the base sequence. 4. Universal: except in a few instances, all organisms use the same code. The Genetic Code (DNA) All 64 codons have meaning; 61 code for an AA and three code for the “stop” signal. Multiple codes for an AA tend to have two bases in common. E. g. CUU, CUC, CUA, CUG code for leu (codons are written: 5’-> 3’ sequence.) A complete table for the genetic code follows on the next slide. G e n e t i Gene Regulation Gene regulation: the various methods used by organisms to control which genes will be expressed and when. – Some regulations operate at the transcriptional level (DNA -> RNA) – Others operate at the translational level (mRNA -> protein). Gene Regulation: Transcriptional Level In eukaryotes, transcription is regulated by three elements: promoters, enhancers, and response elements. Promoters – located adjacent to the transcription site. – are defined by an initiator and conserved sequences such as TATA or GC boxes. – Different transcription factors bind to different modules of the promoter. – Transcription factors allow the rate of synthesis of mRNA (and from there the target protein) to vary by a factor of up to a million. Promoters – Transcription factors find their targeted sites by twisting their protein chains so that a certain amino acid sequence is present at the surface. – One such conformational twist is provided by metal-binding fingers (next screen). – Two other prominent transcription factor conformations are the helix-turn-helix and the leucine zipper. – Transcription factors also possess repressors, which reduce the rate of transcription. Promoters A schematic of a metal-binding finger. Enhancers Enhancers speed up transcription: – They may be several thousand nucleotides away from the transcription site; a loop in DNA brings the enhancer to the initiation site. Response Elements Response elements are activated by their transcription factors in response to an outside stimulus. – The stimulus may be heat shock, heavy metal toxicity, or a hormonal signal. – The response element of steroids is in front of and about 250 base pairs upstream from the starting point of transcription. Translational Level During translation, there are a number of mechanisms that ensure quality control. AARS control – Each amino acid must bond to the proper tRNA. – Enzymes called aminoacyl-tRNA synthase (AARS) catalyze this bonding. – Each AARS recognizes its tRNA by specific nucleotide sequences. – Further, the active site of each AARS has two sieving sites. Translational Level Termination control – The stop codons must be recognized by release factors. – The release factor combines with GTP and binds to the ribosomal A site when that site is occupied by the termination codon. Post-translational control – In most proteins, the Met at the N-terminal end is removed by Met-aminopeptidase. – Certain proteins called chaperones help newly synthesized proteins to fold properly. – If rescue by chaperones fails, proteasomes may degrade the misfolded protein. Recombinant DNA Restriction enzymes are bacterial enzymes that cut the backbone of DNA at specific nucleotide sequences. Donor and plasmid (bacteria) DNA are cleaved by the same restriction enzyme. Donor and plasmid DNA are mixed and donor fragment joins to a complimentary plasmid fragment due to hydrogen bonding. Plasmid ring is restored using DNA ligase. Engineered plasmid (recombinent DNA) is introduced to a bacterium to be reproduced. Polymerase Chain Reaction (PCR) DNA is mixed with Taq polymerase (a heat stable DNA polymerase), a primer DNA sequence for a specific gene, and the four nucleotide triphosphates. A thermocycler raises the temperature to 94- 96 oC to separate the DNA strands, lowers the temperature to 50-56 oC to the primers to hybridize to the DNA, and raises the temperature to 72 oC to allow the Taq polymerase to act. Repeating the cycle doubles the new DNA strands each cycle. (1→2→4→8→16→32 etc) PCR: Heating and Reaction The subject DNA is heated (to separate strands) with – Taq polymerase (enyzme) and Mg2+ – Deoxynucleotide triphosphates – Two, oligonucleotide primers, each complementary to the sequence at the end of one of the target DNA segments PCR: Annealing and Growing Temperature is reduced to 37 to 50°C, allowing the primers to form H-bonds to their complementary sequence at the end of each target strand PCR: Taq Polymerase The temperature is then raised to 72°C, and Taq polymerase catalyzes the addition of further nucleotides to the two primed DNA strands PCR: Growing More Chains Repeating the denature–anneal–synthesize cycle a second time yields four DNA copies, a third time yields eight copies, in an exponential series. PCR has been automated, and 30 or so cycles can be carried out in an hour.