Genes and How They Work Chapter 15

The Nature of Genes Early ideas to explain how genes work came from studying human diseases Archibald Garrod – 1902 Recognized that alkaptonuria is inherited via a recessive allele Proposed that patients with the disease lacked a particular enzyme These ideas connected genes to enzymes

Beadle and Tatum – 1941 Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses Studied Neurospora crassa Used X-rays to damage DNA Looked for nutritional mutations Had to have minimal media supplemented to grow

Beadle and Tatum looked for fungal cells lacking specific enzymes The enzymes were required for the biochemical pathway producing the amino acid arginine They identified mutants deficient in each enzyme of the pathway One-gene/one-enzyme hypothesis has been modified to one-gene/one-polypeptide hypothesis

Central Dogma First described by Francis Crick Information only flows from DNA → RNA → protein Transcription = DNA → RNA Translation = RNA → protein Retroviruses violate this order using reverse transcriptase to convert their RNA genome into DNA

RNA All synthesized from DNA template by transcription Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA) Signal recognition particle RNA Micro-RNA (miRNA) 7

Genetic Code DNA encoded amino acid order Codon – block of 3 DNA nucleotides corresponding to an amino acid Introduced single nulcleotide insertions or deletions and looked for mutations ---Frameshift mutations Indicates importance of reading frame

Marshall Nirenberg identified the codons that specify each amino acid Code is degenerate, meaning that some amino acids are specified by more than one codon

Code practically universal Strongest evidence that all living things share common ancestry Advances in genetic engineering Mitochondria and chloroplasts have some differences in “stop” signals

Prokaryotic transcription Single RNA polymerase Initiation of mRNA synthesis does not require a primer Requires Promoter Start site Transcription unit Termination site

Promoter Forms a recognition and binding site for the RNA polymerase Found upstream of the start site Not transcribed Asymmetrical – indicate site of initiation and direction of transcription

Prokaryotic RNA polymerase Upstream Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. TATAAT– Promoter (–10 sequence)   Holoenzyme Core enzyme Downstream  5׳ ’ 3׳  Start site (+1) Template strand TTGACA–Promoter (–35 sequence) Coding strand Prokaryotic RNA polymerase Upstream 5׳ 3׳ a. b. 13

Recognize specific signal in DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.  binds to DNA  TATAAT– Promoter (– 10 sequence)  Core enzyme Holoenzyme  Downstream 5׳ 5׳ 9 3׳ 3׳  Core enzyme Start site (+1) σ TTGACA – Promoter (–35 sequence) Helix opens at – 1 0 se q uence Template strand Coding strand Prokaryotic RNA polymerase Upstream 5׳ 3׳ 5׳ 3׳ a. b. 14

Initiation 15  TATAAT– Promoter (–10 sequence)  Holoenzyme Core Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.  TATAAT– Promoter (–10 sequence)  Holoenzyme Core enzyme  Downstream 5׳ 9 3׳  Start site (+1) Template strand TTGACA–Promoter (–35 sequence) Coding strand Prokaryotic RNA polymerase Upstream 5׳ 3׳ a. b. Initiation  binds to DNA RNA polymerase bound to unwound DNA Transcription bubble 5׳ 3׳  dissociates ATP Helix opens at –10 sequence Start site RNA synthesis begins 5׳ 3׳ 15

Elongation Transcription bubble – contains RNA polymerase, DNA template, and growing RNA transcript

Termination Terminator sequences: G-C base-pairs --- A-T base-pairs-  phosphodiester bonds (RNA-DNA) in the GC regions called hairpin, (RNAP stop)  4 or ＞4 U (A-U is the weakest of the 4 hybrid base pairs, cannot hold the hybrid strands)  RNA dissociates from the DNA Signal hairpin AU release

Prokaryotic transcription is coupled to translation Operon 1. Grouping of functionally related genes 2. Multiple enzymes for a pathway can be regulated together

Eukaryotic Transcription 3 different RNA polymerases RNA polymerase I transcribes rRNA RNA polymerase II transcribes mRNA and some snRNA RNA polymerase III transcribes tRNA and some other small RNAs Each RNA polymerase recognizes its own promoter

Initiation of transcription Requires a series of transcription factors Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression Interact with RNA polymerase to form initiation complex at promoter Termination Termination sites not as well defined

Eukaryotic Transcription 3 different RNA polymerases RNA polymerase I : rRNA 2.RNA polymerase II: mRNA and some snRNA 3.RNA polymerase III : tRNA and some other small RNAs Each RNA polymerase recognizes its own promoter Other transcription factors Eukaryotic DNA Transcription factor TATA box

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23 Other transcription factors RNA polymerase II Eukaryotic DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Other transcription factors RNA polymerase II Eukaryotic DNA Transcription factor Initiation complex TATA box 23

mRNA modifications In eukaryotes, the primary transcript must be modified to become mature mRNA Addition of a 5′ cap Protects from degradation; involved in translation initiation Addition of a 3′ poly-A tail Created by poly-A polymerase; protection from degradation Removal of non-coding sequences (introns) Pre-mRNA splicing done by spliceosome

5´ cap HO OH P P P CH2 + 3´ poly-A tail 3´ N+ A A A A A A A CH3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ cap HO OH P P P CH2 + 3´ poly-A tail 3´ N+ A A A A A A A CH3 Methyl group A A U A A A mRNA P P P G 5´ CH3

Eukaryotic pre-mRNA splicing Introns – non-coding sequences Exons – sequences that will be translated Small ribonucleoprotein particles (snRNPs) recognize the intron–exon boundaries snRNPs cluster with other proteins to form spliceosome Responsible for removing introns

b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. E1 I1 E2 I2 E3 I3 E4 I4 DNA template Exons Transcription Introns 5׳ cap 3׳ poly-A tail Primary RNA transcript Introns are removed 5׳ cap 3׳ poly-A tail a. Mature mRNA Intron 1 mRNA 3 2 4 DNA 7 5 6 Exon b. c. 27 b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine

2. snRNPs associate with other factors to form spliceosome. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNA snRNPs Exon 1 Intron Exon 2 A 5´ 3´ Branch point A 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. Spliceosome A 5´ 3´ 2. snRNPs associate with other factors to form spliceosome. Lariat A 5´ 3´ 3. 5´ end of intron is removed and forms bond at branch site, forming a lariat. The 3´ end of the intron is then cut. Excised intron Exon 1 Exon 2 5´ Mature mRNA 3´ 28 4. Exons are joined; spliceosome disassembles.

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Alternative splicing Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons 15% of known human genetic disorders are due to altered splicing 35 to 59% of human genes exhibit some form of alternative splicing Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs

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