(genotype, transcription, and translation) From gene to phenotype (genotype, transcription, and translation)

DNA Form & Function

DNA – an ideal genetic material Faithful replication Information content Capable of change

DNA Five carbon sugar – deoxyribose Phosphate group One of four bases Purines Five carbon sugar – deoxyribose Phosphate group One of four bases Pyrmidines http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html

Nucleotide + Nucleotide + Nucleotide + …… DNA: Polymerization Nucleotide + Nucleotide + Nucleotide + …… http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html

Faithful replication Complementary base pairing and the double helix Semi-conservative replication

DNA replication DNTPs: dATP, dGTP, dTTP, and dCTP Template DNA (a pre-existing single strand) DNA polymerase http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleotides.html

Template DNA (a pre-existing single strand) DNA replication Template DNA (a pre-existing single strand)

DNA replication DNA Polymerases Polymerases catalyze the formation of a phosphodiester bond between the 3'-OH of the deoxyribose on the last nucleotide and the 5' phosphate of the dNTP precursor

Replication is 5’ to 3’

Replication is 5’ to 3’

Replication is 5’ to 3’

Replication is 5’ to 3’

Replication is 5’ to 3’

Replication is 5’ to 3’

Information content Met Leu Gly Asp Tyr Gly Phe Lys ATG CTG GGA GAT TAT GGC TTT AAG AUG CUG GGA GAU UAU GGC UUU AAG

Capable of change Replication errors and proofreading Exonuclease activity - removal of unpaired nucleotide from the 3' end of the chain

Mutations result from heritable changes in DNA sequence May affect transcription, translation, and phenotype Single nucleotide substitutions can have different consequences on phenotype Silent *** CTG GGA GAT TAT GGC TTT AAG*** *** CTG GGA GAT TAT GGC TTC AAG*** alignment Leu Gly Asp Tyr Gly Phe Lys Leu Gly Asp Tyr Gly Phe Lys translation Missense *** CTG GGA GAT TAT GGC TAT AAG*** alignment Leu Gly Asp Tyr Gly Tyr Lys translation Nonsense *** CTG GGA GAT TAG GGC TTT AAG*** alignment Leu Gly Asp Tyr Gly Phe Lys Leu Gly Asp STOP translation

DNA Replication - in vivo Replication begins at an origin - proceeds bi-directionally In a higher plant chromosome - thousands of origins The size of the genome The rate of DNA replication The length of the S phase  

Development of the female gametophyte Megaspore mother cell (MMC)  MMC undergoes meiosis Of 4 megaspores produced 1 survives (most species) Three post-meiotic mitoses   1 2 3

Development of the male gametophyte The first mitosis gives vegetative and generative nuclei; at the second mitotic division, the generative nucleus gives 2 sperms. mitosis mitosis mitosis mitosis mitosis mitosis mitosis mitosis

Replication details Unwinding: The DNA helix opened by helicase enzymes, which break the hydrogen bonds holding the two strands of the helix together. Gyrase facilitates helicase action by relieving tension in coiled DNA. Stabilization: The unwound DNA is stabilized by a protein (single strand binding protein (SSB)), which speeds up DNA replication. http://www.dnareplication.info/stepsofdnareplication.php

Replication details Priming: Primases form short RNA primers Synthesis: DNA polymerases use the primer to synthesize the new strand (polymerases cannot start synthesis on their own) Primer removal: RNA primers removed by exonuclease activity of a polymerase and replaced with DNA http://www.dnareplication.info/stepsofdnareplication.php

Replication details DNA polymerases synthesize new strands 5’ to 3’ The double helix is antiparallel DNA synthesis is semi-conservative Therefore DNA synthesis is continuous on the leading strand and discontinuous on the lagging strand Multiple priming sites on the lagging strand - Okazaki fragments http://www.dnareplication.info/stepsofdnareplication.php

Replication details DNA Pol I exonuclease removes RNA Primers in lagging strand DNA Polymerase adds complementary nucleotides to fill the gaps DNA Ligase adds phosphate in the remaining gaps of the phosphate - sugar backbone

Replication details Specific polymerases with 3’- 5’ exonuclease activity identify and remove detected mis-matches on the synthesized strand Proofreading starts at the 3’ end of the synthesized strand and proceeds 3’ - 5’

DNA to RNA to protein

From gene to phenotype “Central dogma” DNA → RNA → protein DNA Covered barley: Lemma and palea adhere to seed RNA Other genes Pathways Environmental signals Protein

Eukaryotic Gene Structure “DNA sequence specifying a protein” ~200 – 2,000,000 nt (bp)

RNA Ribonucleic acid (RNA) A key nucleic acid in transcription, translation, and regulation Like DNA except that: Usually single rather than double stranded Pentose sugar is ribose rather than deoxyribose It contains the pyrimidine base uracil (U) rather than thymine (T)     

Classes of RNA Informational (messenger): mRNA - Transcription Functional (transfer, ribosomal RNA): tRNA, rRNA - Translation Regulatory (addressed later in the context of gene regulation)

Informational (messenger) - mRNA Single-stranded RNA molecule complementary to one of the DNA strands of a gene Leaves nucleus; in cytoplasm translated into protein http://www.genome.gov/glossary

Functional (transfer) - tRNA Molecules that carry amino acids to the growing polypeptide: ~ 32 different kinds of tRNA in a typical eukaryotic cell Each the product of a separate gene ~ 80 nucleotides Double and single stranded regions Unpaired regions form loops

Functional (transfer) - tRNA Each kind of tRNA carries (at its 3′ end) one of the 20 amino acids  At one loop, 3 unpaired bases form an anticodon Base pairing between the anticodon and the complementary codon on a mRNA molecule brings the correct amino acid into the growing polypeptide chain

Functional (ribosomal) - rRNA The ribosome consists of RNA and protein Site of protein synthesis Translation of mRNA sequence

Transcription The template strand is also referred to as the antisense (or non-coding)  strand and the non-template strand as the sense (or coding) strand 

Transcription Either strand of the DNA may be the template strand for RNA synthesis for a given gene 

Transcription: Initiation Initiation: Transcription initiated at the promoter Promoters Control gene expression: rate, constitutive, inducible Identify by consensus sequences

Transcription: Elongation

Transcription: Elongation

Transcription: Elongation

Transcription: Elongation

Transcription: Elongation

Transcription: Termination Specific nucleotide sequence in the DNA signals end of transcription: the “terminator” sequence The terminator sequences recognized by specific proteins associated with RNA polymerase II Signal end of transcription and addition of poly-A tail

Transcript Processing Eukaryotes - primary RNA transcript is processed into a mature mRNA before export to the cytoplasm for translation

Transcript Processing 5’ cap: 7-methylguanosine added to free phosphate at 5’ mRNA Prevents degradation and assists in ribosome assembly 3’poly(A tail): After pre-mRNA is cleaved, poly (A) polymerase adds ~200 A nucleotides Protects against degradation, aids export to cytoplasm, and involved in translation initiation Splicing: Removal internal portions of the pre-mRNA Most eukaryotic genes have an intron/exon structure Splicing removes introns and remaining exons are rejoined

Plant Gene Structure

The Code 64 codons; 61 represent amino acid codes and 3 cause the termination of protein synthesis (stop codons) Degeneracy: Most amino acids represented by >1 codon

Translation MDTVAAWPQFEEQDYMTVWPEEQEYRTVWSEPPKRRAGRIKLQETRHPVYRGVRRRGKVGQWVCELRVPVSRGYSRLWLGTFANPEMAARAHDSAALALSGHDACLNFADSAWRMMPVHATGSFRLAPAQEIKDAVAVALEVFQGQHPADACTAEESTTPITSSDLSGLDDEHWIGGMDAGSYYASLAQGMLMEPPAAGGWREDDGEHDDGFNTSASLWSY

Translation – Initiation

Translation – Elongation

Translation – Termination

gene            <1..>77 /gene="CBF2A" mRNA          <1..>772     /gene="CBF2A"                  /product="HvCBF2A” 5'UTR          <1..12      CDS            13..678      /gene="CBF2A”                  /note="HvCBF2A-Dt; AP2 domain CBF protein; putative CRT         binding factor; monocot HvCBF4-subgroup member                   /codon_start=1                   /product="HvCBF2A"   /protein_id="AAX23688.1" /db_xref="GI:60547429"                     /translation="MDTVAAWPQFEEQDYMTVWPEEQEYRTVWSEPPKRRAGRIKLQE TRHPVYRGVRRRGKVGQWVCELRVPVSRGYSRLWLGTFANPEMAARAHDSAALALSGH DACLNFADSAWRMMPVHATGSFRLAPAQEIKDAVAVALEVFQGQHPADACTAEESTTP  ITSSDLSGLDDEHWIGGMDAGSYYASLAQGMLMEPPAAGGWREDDGEHDDGFNTSASL  WSY"     3'UTR          679..>772                    

Summary of Transcription and Translation Sequence Type 5' atg gac aca.........tag 3’ Sense (Non-template DNA) (decode replacing T with U ) 3' tac ctg  tgt.........atc 5' Anti-sense (Template DNA) 5'aug gac aca........uag3' RNA (decode) M     D     T           Stop Amino acid code (See Table) Methionine, Aspartic acid, Threonine

Amino Acid Abbreviations

Proteins Primary, secondary, tertiary, and quaternary structures

Structural proteins: economic and health implications Wheat endosperm storage proteins feed millions and can cause intense suffering in individuals with celiac disease

Transcription, Translation, Phenotype Fragrance in rice