DNA structure and synthesis

1. DNA structure and synthesis nucleotides • DNA is a polymer of

2. Nucleotide base terminology Nucleotides generally have 1 (mono), 2 (di) or 3 (tri) phosphate groups. Nucleosides that make up DNA have as sugar; have deoxy as a prefix in the name. deoxyribose instead of ribose

3. Which bases pair with each other?

4. DNA “backbones”: alternating sugar-phosphates. Interior: Complemen-tary bases. T always pairs with A, C with G. DNA chains are antiparallel. http://courses.bio.psu.edu/fall2005/biol230weve/tutorials/tutorial2_files/figure_16_5_part2.gif

5. directionality in DNA

6. DNA chains held together with H bonds. A-T pairs: 2 G-C pairs: 3. Bases are flat, planar; they stack on the inside of the molecule. Hydrophobic interactions stabilize DNA. DNA chains twist together around http://genetics.nbii.gov/images/BasePairs.gif a central axis, not around each other.

7. Structure/Function Relationships in DNA • Note how the structure presents a mechanism for exact replication, needed for the genetic molecule. • Bases can be arranged in any sequence; provides info for specifying 20 amino acids. • Mispairing due to mistakes, damage, lead to mutation, lead to individual variation and evolution.

8. “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." -Watson and Crick http://www.sciencetechnologyaction.com/lessons2.php?studyid=6&edition=1

9. DNA replication • Origin of DNA replication: particular site on DNA where copying of the DNA always starts. • Replication is bidirectional • In each direction, there is a replication fork. • Most bacterial DNA is circular, so there is one Origin and one terminus • Replicon: a length of DNA molecule replicated after initiation from one origin. Examples: • Bacterial DNA, plasmids, segments of eukaryotic chromosomes.

10. E. coli, a typical impatient bacterium • E. coli takes 30 minutes to replicate all its DNA, yet it can double every 20 minutes. How does it do this? • Starts a round of DNA replication before finishing the previous round.

11. A couple of words on terminology • A chemical reaction in which molecules are combined to make a products is a synthesis reaction. • DNA is synthesized in cells, but we can direct DNA synthesis in a test tube also. PCR, sequencing both involve DNA synthesis. • DNA replication is a natural biological process in which a DNA molecule is copied in a cell. • Replication is a specific act of synthesis.

12. What every DNA polymerase needs • A template of DNA • Enzymes copy a single strand of DNA • Can’t work without something to copy from • A primer • A primer is a polynucleotide with a “free 3´OH end” • In normal DNA replication, this is RNA • A substrate • To make DNA, a polymer, monomers are needed • Nucleotide triphosphates (NTPs) are the monomers

13. Numbering of ring positions Ring positions on nitrogenous bases “use up” the numbers, so positions on sugar are indicated by “prime”. 5’ and 3’ positions on sugar are very important.

14. Adding and removing bases:Directionality • DNA synthesis is ALWAYS in a 5´ to 3´ direction • See next slide. • All 3 DNA pols have a 3´ to 5´ exonuclease activity • Nuclease: enzyme activity that cuts nucleic acids • Exo- means cuts from an end • 3´ to 5´ means the opposite direction from synthesis • “proofreading” ability; polymerase can “backspace” to remove a base put it by mistake. • DNA pol I has a 5´ to 3´ exonuclease activity • Cuts off DNA bases in same direction as synthesis

15. * * * *

16. Initiation of DNA replication * Helicases unwind the DNA * • DNA pol requires a primer to add to: Primase makes an RNA • Synthesis is 5´ to 3´, and antiparallel. • Leading strand; synthesis follows replication fork.

17. Problems due to antiparallel nature of DNA In this picture, replication of the lower strand of DNA can proceed as the “replication fork” moves from right to left because the direction of synthesis of new DNA is 5’ to 3’. What about the other strand? The one made without a hitch is called the “leading strand”, the other is the “lagging strand”.

18. Okazaki fragments Because of requirement for 5’to 3’ synthesis, lagging strand must repeatedly top and start; needs an RNA primer each time.

19. Cleaning up Okazaki’s Ligase needed DNA Pol I cuts out RNA primers, replaces them with DNA. Uses both the 5´ to 3´ exonuclease and polymerase activities.

20. About RNA 1) DNA is double stranded, but RNA is single stranded. However, RNA can base-pair with itself to create double stranded regions. RNA DNA tRNA genetics.gsk.com/graphics/ dna-big.gifhttp://www.fhi-berlin.mpg.de/th/JG/RNA.jpg http://www.santafe.edu/images/rna.gif

21. About RNA-2 2) RNA contains ribose instead of deoxyribose 3) RNA contains uracil instead of thymine. www.layevangelism.com/.../ deoxyribose.htmhttp://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/images/uracil.gif

22. 3 kinds of RNA mRNA: a copy of the gene; is translated to make protein. tRNA: smallest RNA, does actual decoding. rRNA: 3 sizes that, along with proteins, make up a ribosome. tRNA rRNA http://www.cu.lu/labext/rcms/cppe/traducti/tjpeg/trna.jpeg; Tobin and Duschek, Asking About Life; http://www.tokyo-ed.ac.jp/genet/mutation/nort.gif

23. Transcription: making mRNA • RNA a polymer assembled from monomers • Ribonucleoside triphosphates: ATP, UTP, GTP,CTP • RNA polymerase • Multi-component enzyme • Needs a template, but NOT a primer • In bacteria, a component (sigma) recognizes the promoter as the place on DNA to start synthesis • Synthesis proceeds 5’ to 3’, just as in DNA • mRNA is complementary and antiparallel to the DNA strand being copied.

24. Transcription-2 • The order of nucleotides in the RNA reflects the order in the DNA • If RNA is complementary to one DNA strand, then it is identical (except for T change to U) to the other DNA strand. Either DNA strand may contain the gene! Transcription just runs the other direction.

25. Sense, antisense Compare the sense strand of the DNA to the mRNA. Note that mRNA synthesis will be 5’ to 3’ and antiparallel. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/S/SenseStrand.gif

26. Transcription needs a Promoter A promoter is non-transcribed DNA Prokaryotes Eukaryotes http://opbs.okstate.edu/~petracek/2002%20Gene%20expression/img043.gif

27. The Process of Transcription • Promoter recognition: 2 consensus sequences • The -10 region: TATAAT (10 bases upstream from where transcription actually starts. • The -35 region, farther upstream, also important. • “Consensus” sequence meaning the DNA sequence from many genes averages out to this. • The closer these 2 regions actually are to the consensus sequences, the “stronger” the promoter, meaning the more likely RNA polymerase binding and transcription will occur.

28. Consensus sequence Numbers indicate the percentage of different genes in which that nucleotide appears in that spot in the promoter sequence. http://www.uark.edu/campus-resources/mivey/m4233/promoter.gif

29. The Process of Transcription-2 • After binding to the promoter, polymerase “melts” DNA, lines up first base at the +1 site = Initiation. • RNA synthesis continues (Elongation), only the template strand being transcribed. • Termination: must be a stop sign, right? • In bacteria, hairpin loop followed by run of U’s in the RNA. Of course, the DNA must code for complementary bases and a run of A’s. See next. • Termination factor “rho”. Accessory protein.

30. Termination of Transcription in Bacteria The hairpin loop destabilizes the interactions between the DNA, mRNA, and polymerase; U-A basepairs are very weak, and the complex falls apart. In euks, termination occurs with a processing step. http://www.blc.arizona.edu/marty/411/Modules/Weaver/Chap6/Fig.0649ac.gif

31. About mRNA structure, etc. • Start site of transcription is NOT equal to start site of Translation • First codon read, AUG, is downstream from the first ribonucleotides. +1 is transcription start, not translation start. • AUG marks the beginning of an Open Reading Frame (ORF). • Lifetime of a eukaryotic mRNA is variable • For prokaryotes, mRNA is short lived, fits in with need of microbes to respond quickly to changes in environment.

32. Storage and use of genetic information • The genetic code • Three bases (in a row) specify an amino acid • Transcription • The synthesis of a mRNA, complementary to one of the DNA strands, containing the genetic code • Translation • Proteins and rRNAs in the ribosome along with tRNAs translate the genetic code into proteins. • Post-translational modification • Proteins are altered after synthesis

33. The Genetic Code • Four bases taken how many at a time? Need to code for 20 different amino acids. • Each base = 1 amino acid: only 4 • Every 2 bases = 1 a.a.: 16 combinations, 4 short. • Every 3 bases: 64 combinations, enough. • Every 3 bases of RNA nucleotides: codon • Each codon is complementary to 3 bases in one strand of DNA • Each codon (except for T →U switch) is the same as 3 bases in the other DNA strand.

34. More about the Genetic Code • The code is • Unambiguous: each codon specifies 1 amino acid • Degenerate: a particular amino acid can be coded for by several different codons. • Ordered: similar codons specify the same amino acid. • Commaless, spaceless, and non-overlapping : each 3 bases is read one after the other. • Punctuated: certain codons specify “start” and “stop”. • Universal: by viruses, both prokaryotic domains, and eukaryotes (except for some protozoa, mitochondria).

35. The Genetic Code-2 http://www.biology.arizona.edu/molecular_bio/problem_sets/nucleic_acids/graphics/gencode.gif

36. Wobble • Crick’s Wobble Hypothesis • The code is “ordered” • The first 2 positions are more important • When lining up with the anticodon of the tRNA, the third position doesn’t bind as tightly, thus a looser match is possible. • Because of this flexibility, a cell doesn’t need 61 different tRNAs (one for each codon). • Bacteria have 30-40 different tRNAs • Plants, animals have up to 50.

37. tRNA 3D structure: The familiar loops of the 2D structure are labeled. 3’ end: Attaches to amino acid. Decoder end: Complementary to codon. hto-b.usc.edu/~cbmp/2001/ tRNA/trna%20s1.jpg

38. Translation • mRNA: provides message to be translated. • Ribosomes: functional workbench for synthesis. • tRNA: bring aa to ribosome, decode mRNA. • Aminoacyl tRNA synthetases: enzymes that attach amino acids to tRNAs. • Protein factors: help move process along: initiation, elongation, and termination. • Process is similar, but different between prokaryotes and eukaryotes.

39. Initiation and Termination of protein synthesis • AUG is always the first codon (initiator codon) • Establishes an “open reading frame” (ORF) • Ribosome begins synthesis with a methionine • In bacteria, it is N-formylmethionine (fMet) • After synthesis , either formyl group is removed or entire fMet is removed (Met in eukaryotes) • Three codons serve as termination codons: • UGA, UAG, UAA; any one can be a stop signal • Do NOT code for an amino acid • Cause translation to end; protein is completed formyl

40. Translation-1 • Initiation • Small subunit, mRNA, met-tRNA, IFs, GTP • mRNA: sequence for binding to ribosome needed • prokaryotes: Shine-Delgarno • Eukaryotes: Cap and Kozak sequence • (GCC)RCCATGG where R is a purine • First tRNA is fMet-tRNA in prokaryotes • IFs are protein Initiation Factors • GTP needed for energy • When all have come together, Large subunit added

41. Translation-2 • Ribosome has 3 sites • AA site where tRNA-aa first sits in • P site where tRNA with growing peptide sits • E or Exit, site transiently occupied by used tRNA • Elongation, with help of EFs and GTP • tRNA with new aa sits in A site • Stays in A site if anticodon on tRNA is complementary to codon on mRNA. • tRNA in P site transfers growing chain to new aa • Catalyzed by rRNA • Ribosome moves relative to mRNA and tRNAs • tRNAs now in new sites, new codon lined up

42. Ribosome schematic http://staff.jccc.net/pdecell/proteinsynthesis/translation/elongation12.gif

43. Translation-3 • Termination • When stop codon is in A site, no tRNA binds • GTP-dependent release factor (protein) removes polypeptide from tRNA in P site. All done. • Ribosomal subunits typically dissociate. • Do a Google Search for translation animation • Many hits. Note presence, absence of E site • Note shape of ribosomes • Note whether role of rRNA in catalysis is shown

44. Nonsense mutations and suppressors • A mutation may change a normal codon to a stop codon; protein synthesis ends prematurely. (nonsense mutation) • A second mutation can cure the original: a “suppressor”. • If the gene for a tRNA is mutated in the anticodon so that the stop codon is now read by the tRNA.

45. Polysomes and Polycistronic mRNA • In eukaryotes, when mRNA enters the cytoplasm, many ribosomes attach to begin translation. A mRNA w/ many ribosomes attached = polysome. • In eukaryotes, the mRNA for a single gene is processed and translated; in prokaryotes, mRNA can be polycistronic, meaning several genes are on the same mRNA and are translated together • With no nucleus, translation can begin in prokaryotes before transcription is over.

46. Polysomes Multiple ribosomes attach to the mRNA and begin translating. Strings of ribosomes can be seen attached to the mRNA. http://opbs.okstate.edu/~petracek/Chapter%2027%20Figures/Fig%2027-29b-bottom.GIF www.cu.lu/labext/rcms/ cppe/traducti/tpoly.html