Nucleic Acid

DNA RNA protein The Central Dogma Replication Transcription Translation The flow of genetic information is unidirectional, from DNA to protein with messenger RNA as an intermediate.

DNA Replication - the process of making new copies of the DNA molecules Potential mechanisms: organization of DNA strands Conservative old/old + new/new Semiconservative old/new + new/old Dispersive mixed old and new on each strand

Meselson and Stahl’s replication experiment

Replication as a process 1. Double-stranded DNA unwinds. 2. The junction of the unwound molecules is a replication fork. 3. A new strand is formed by pairing complementary bases with the old strand. 4. Two molecules are made. Each has one new and one old DNA strand.

Enzymes in DNA replication Primase adds short primer to template strand Helicase unwinds parental double helix Binding proteins stabilize separate strands DNA polymerase binds nucleotides to form new strands Exonuclease removes RNA primer and inserts the correct bases Ligase joins Okazaki fragments and seals other nicks in sugar-phosphate backbone

Replication Helicase protein binds to DNA sequences called Primase protein makes a short segment of RNA complementary to the DNA, a primer. 5’ 3’ Binding proteins prevent single strands from rewinding. 3’ 5’ Helicase protein binds to DNA sequences called origins and unwinds DNA strands.

Replication DNA polymerase enzyme adds DNA nucleotides Overall direction of replication 5’ 3’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer.

Replication Leading strand synthesis continues in a 5’ 3’ Overall direction of replication Leading strand synthesis continues in a 5’ to 3’ direction.

Replication Leading strand synthesis continues in a 3’ 5’ Overall direction of replication Okazaki fragment Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication Leading strand synthesis continues in a Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication Leading strand synthesis continues in a 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication Leading strand synthesis continues in a 5’ 3’ 3’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.

Replication 3’ 5’ 5’ 5’ 3’ Exonuclease enzymes remove RNA primers.

Replication Exonuclease enzymes remove RNA primers. 3’ 5’ Ligase forms bonds between sugar-phosphate backbone. Exonuclease enzymes remove RNA primers.

Replication 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’

Transcription

Prokaryotic Gene Structure Promoter CDS Terminator UTR UTR Genomic DNA transcription mRNA translation protein

Eukaryotic Gene Structure 5’ - Promoter Exon1 Intron1 Exon2 Terminator – 3’ UTR splice splice UTR transcription Poly A translation protein

Transcription initiation and elongation 1. Genes need to be expressed to be “genes” 2. Transcription is directed to specific locations (promoters) 3. RNA is elongated in the 5’-to-3’ direction

Promoter Promoter determines: Which strand will serve as a template. Transcription starting point. Strength of polymerase binding. Frequency of polymerase binding.

Prokaryotic Promoter One type of RNA polymerase. Pribnow box located at –10 (6-7bp) –35 sequence located at -35 (6bp)

Eukaryote Promoter 3 types of RNA polymerases are employed in transcription of genes: RNA polymerase I transcribes rRNA RNA polymerase II transcribes all genes coding for polypeptides RNA polymerase III transcribes small cytoplasmatic RNA, such as tRNA.

Eukaryote Promoter Goldberg-Hogness or TATA located at –30 Additional regions at –100 and at –200 Possible distant regions acting as enhancers or silencers (even more than 50 kb).

RNA Synthesis DNA template: 3’-to-5’ RNA synthesis: 5’-3’; no primer needed

Termination Sites The newly synthesized mRNA forms a stem and loop structure (lollipop). A disassociation signal at the end of the gene that stops elongating and releases RNA polymerase. All terminators (eukaryotes and prokaryotes) form a secondary structure.

Termination Sites The terminator region pauses the polymerase and causes disassociation.

Splice Sites Eukaryotics only Removing internal parts of the newly transcribed RNA. Takes place in the cell nucleus (hnRNA)

Splice Sites Conserved splice sites are shared by both the exon and the intron. Different signals on the donor site (3’) and on the acceptor site (5’).

Translation

Genetic Terminology Chromosome - threadlike structures in the nucleus that carry genetic information Gene - fundamental unit of heredity - inherited determinant of a phenotype - sequence of DNA that instructs a cell to produce a particular protein DNA - deoxyribonucleic acid, - the genetic material - the biochemical that forms genes

Open Reading Frames (ORF)

TRANSCRIPTION TRANSLATION Unwinding of gene regions of a DNA molecule Pre mRNA Transcript Processing mRNA rRNA tRNA protein subunits Mature mRNA transcripts ribosomal subunits mature tRNA Convergence of RNAs TRANSLATION Cytoplasmic pools of amino acids, tRNAs, and ribosomal subunits Synthesis of a polypetide chain at binding sites for mRNA and tRNA on the surface of an intact ribosome FINAL PROTEIN Destined for use in cell or for transport

PROTEIN TRANSLATION m-RNA GOES THRU RIBOSOME. RIBOSOME IS r-RNA,CODE THREADS THRU RIBOSOME. AREA OF RIBOSOME BOUND TO tRNA 20 TYPES OF AA ANTICODON ON ONE END OF t-RNA. AA ON OTHER END OF t-RNA AA ATTACH TO EACH OTHER IN PEPTIDE BOND FORM PROTEINS

The triplet code

Role of Ribosome 70S ribosome 50S subunit 30S subunit 16S rRNA 34 proteins 21 proteins 23S rRNA 5S rRNA

mRNA  Ribosome mRNA leaves the nucleus via nuclear pores. Ribosome has 3 binding sites for tRNAs: A-site: position that aminoacyl-tRNA molecule binds to vacant site P-site: site where the new peptide bond is formed. E-site: the exit site Two subunits join together on a mRNA molecule near the 5’ end. The ribosome will read the codons until AUG is reached and then the initiator tRNA binds to the P-site of the ribosome. Stop codons have tRNA that recognize a signal to stop translation. Release factors bind to the ribosome which cause the peptidyl transferase to catalyze the addition of water to free the molecule and releases the polypeptide.

Purpose of tRNA The proper tRNA is chosen by having the corresponding anticodon for the mRNA’s codon. The tRNA then transfers its aminoacyl group to the growing peptide chain. For example, the tRNA with the anticodon UAC corresponds with the codon AUG and attaches methionine amino acid onto the peptide chain.

tRNAs are specific carriers of amino acids Aminoacyl-tRNA synthetases attach specific amino acids to heat-stable tRNA molecules ATP + Amino acid Amino acid 3’ 5’ aminoacyl-tRNA synthetase tRNA e.g. tRNAPhe aminoacylated tRNA e.g. Phe-tRNAPhe

RNA  Protein: Translation Ribosomes and transfer-RNAs (tRNA) run along the length of the newly synthesized mRNA, decoding one codon at a time to build a growing chain of amino acids (“peptide”) The tRNAs have anti-codons, which complimentarily match the codons of mRNA to know what protein gets added next But first, in eukaryotes, a phenomenon called splicing occurs Introns are non-protein coding regions of the mRNA; exons are the coding regions Introns are removed from the mRNA during splicing so that a functional, valid protein can form

Translation, continued Catalyzed by Ribosome Using two different sites, the Ribosome continually binds tRNA, joins the amino acids together and moves to the next location along the mRNA ~10 codons/second, but multiple translations can occur simultaneously

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