Eukaryotic cells. Molecular Testing and Clinical Diagnosis. Organelles. Common organelles include
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1 Molecular Testing and Clinical Diagnosis Molecular Biology Review Eukaryotic cells Contain internal organelles membrane bound structures separate specific regions from the rest of the cytoplasm include: Fungi Algae Protozoa Plants Animals Common organelles include nucleus smooth and rough endoplasmic reticulum golgi lysosomes, peroxisomes some organisms have chloroplasts Organelles Separate regions with unique function nucleus replication (DNA made into DNA) transcription (DNA made into RNA cytoplasm translation (mrna into Protein) protect components from digestive enzymes in cytoplasm Nucleic Acid Composition Sugar deoxyribose DNA ribose RNA Nucleotide base Adenine, Guanine, Cytosine, Thymine and in RNA, Uracil Phosphate DNA Structure Two strands of nucleic acid in opposite orientation P-sugar backbone on the outside of ladder nucleotide bases in the middle hydrophobic interaction P-sugar bonds are covalent nucleotide base pair bonds are Hydrogen bonds Note negative charge on P groups Student Laboratory Molecular: Molecular Biology Review Page 1
2 DNA strands are held precisely together in opposite orientation based upon sugar bonds involved in Phospho- di-ester bond 5` to 3` on one side 3` to 5` on the other side bases are flipped or opposite Opposite Orientation Allows: Aligns nucleotide bases for hydrogen bonding Hydrogen bonding is : a weak association between an electronegative atom (O or N) and a hydrogen atom the hydrogen atom has an overall positive charge since its electron is utilized in the covalent bond Nucleotides bind specifically Adenine can only align and juxtaposition with thymine or uracil to form 2 hydrogen bonds Cytosine can only align and juxtaposition with guanine to form 3 hydrogen bonds Thermodynamically G-C pairs are stronger that A-T or A-U pairs due to the 3:2 hydrogen bonds Overall Structure of DNA Double helix Opposite orientation Held together by H bonds and hydrophobic interaction of nucleotides A binds T or U only G C only Inherent within the ACGT order of DNA is the specified protein product RNA production DNA made into RNA: transcription Many types of RNA nucelolar RNA heteronuclear RNA mrna ribosomal RNA transfer RNA Nucleolar RNA Within the nucleolus found in the nucleus site of pre-ribosomal RNA (rrna) synthesis allows for rrna cleavage and its association with ribosomal proteins to form ribosomal subunits 40S subunit & 60S subunit Student Laboratory Molecular: Molecular Biology Review Page 2
3 Heteronuclear RNA DNA made into RNA: transcription Newly transcribed mrna is hnrna Transcription is a complex process requiring: double stranded DNA template promoter sequence in the DNA template activator proteins binding at the promoter RNA polymerase enzyme RNA polymerase makes multiple complimentary copies of DNA into hrna synthesizes only in the 5` to 3` direction unwinds DNA by itself binds ribonucleic acid into a strand complimentary to DNA ACGT order mrna Associates with ribosomes mrna translated 3 nucleotides (codon) at a time Start each protein with AUG sequence which signals a methionine open reading frame shifts Ribosomal RNA (rrna) Synthesized, processed and associates with ribosomal proteins in the nucleolus form secondary structure by folding back on itself and binding (H bonds and disulfide) move to cytoplasm as individual units 40S and 60S forms translation initiation complex (80S) in the cytoplasm requires mrna and GTP Transfer RNA (trna) Each amino acid has a unique trna each trna activated (charged with an amino acid) by a specific trna synthase each trna has anticodon sequence complimentary to mrna codon for amino acid specificity trna characteristics Short nucleotides in length RNA sequence always ends in CCA Amino acid attach at the 3` end through a OH group three dimensional structure clover leaf with mini double helices anticodon located in the center of middle loop complimentary to mrna codon Student Laboratory Molecular: Molecular Biology Review Page 3
4 Codon reading mrna made into protein: translation mrna codon CCC trna anticodon GGG third position wobble GGG corresponds to trna carrier for amino acid proline Note AUG signal methionine start and UAA or UAG signal stop Translation initiation 5` cap binds initiation factors in the cytoplasm 40S subunit binds mrna in the presence of GTP and shifts to AUG codon (start) 60S subunit joins to form the initiation complex Translation Initiation complex brings in GTP activated methionine trna initiation complex shifts to second codon, reads and brings in GTP activated trna peptide bond forms ribosome shifts down mrna (translocation) requires GTP hydrolysis continuous translocation and elongation Translocation and elongation GTP charged trna with appropriate amino acids are brought to ribosome as mrna codons are read mrna read until stop codon encountered translocation GTP dependent ribosome mrna complex breaks apart Protein products mrna initiates translation complex with ribosome in cytosol and generates proteins released directly into cytosol enzymes cytoskeletal proteins regulatory proteins Protein products mrna initiates translation complex with the ribosome, protein sequence (signal sequence) generated may signal the need for protective protein production protein made directly into the lumen of ER secretory proteins transmembrane proteins lytic enzymes proteins that require posttranslation modification Student Laboratory Molecular: Molecular Biology Review Page 4
5 Transmembrane proteins Loop back and forth through the membrane while mrna being translated may associate with other transmembrane subunits after synthesis membranes are then pinched off and directed to target membrane or organelles Protein processing Proteins are processed as they pass from the RER through the golgi and directed to their target membranes processing enzymes are present in the ER and golgi cleavage glycosylation disulfide bridging Summary Unique DNA structure and nucleotide binding A binds T or U only G bind C only DNA made into DNA: replication (nucleus) DNA made into RNA: transcription (nucleus) mrna made into protein: translation (cytoplasm) Student Laboratory Molecular: Molecular Biology Review Page 5
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