CHAPTER 8 The Molecular Genetics of Gene Expression Gene expression information contained in genes is decoded to produce other molecules that determine the phenotypic traits of organisms ONE RNA synthesized by RNA polymerase which uses template DNA RNA transcription TWO RNA undergoes chemical modification called RNA processing in nucleus of Euk cells THREE RNA protein translation makes a gene product 8 1 Polypeptide chains are linear polymers of amino acids Each amino acid contains a carbon which is attached to a carboxyl COOH amino NH2 and a side R group chain peptide bond forms between carboxyl of one and amino of another chains Ends of chains are called amino terminus and carboxyl terminus add to CARBOXYL end number from AMINO order in which they are added A protein contains SUBUNITS when it contains more than one polypeptide chain Human proteins and other vertebrates have more complex domain structure than invertebrate A domain is a relatively independent folding unit have specialized function Only 7 of human proteins and domains are specific to vertebrates not found in others 8 2 The linear order of amino acids is encoded in a DNA base sequence Order of nucleotides in a gene order of amino acids in a polypeptide trpA in E coli Colinearity is when the sequence of base pairs in DNA determines the sequence of amino acids in the polypeptide in a collinear point to point manner universal for Prok 8 3 The base sequence in DNA specifies the base sequence in an RNA transcript The chemical synthesis of RNA is similar to that of DNA Precursors in RNA synthesis are four ribonucleoside 5 triphosphates ATP GTP CTP and UTP Sugar phosphate bond forms between 3 hydroxyl group and 5 triphosphate of the next The enzyme used in transcription is RNA polymerase which binds to promoter DNA sequence The transcription start site is a nucleotide near the promoter where transcription begins Unlike DNA polymerase RNA polymerase can initiate chain growth without a primer Eukaryotes have several types of RNA polymerase RNA polymerases are large multi subunit complexes whose active form is RNA polymerase holoenzyme Prok One RNA polymerase six subunits in holoenzyme Processivity of RNA polymerase is HIGH 10 4 for prok and 10 6 for euk Rate of transcription 70 nucleotides s for prok and 40 nucleotides s for euk Euk RNA polymerase I produces transcript that is processed into rRNA transcription start site promoter RNA polymerase II transcribes all protein coding genes and small nuclear RNA genes promoter upstream of transcription start site on 5 side holoenzyme has 12 polypeptide subunits and a mass of 500kD RNA polymerase III transcribes all tRNA genes and 5S component of rRNA promoter downstream of transcription start site on 3 side Promoter recognition typically requires multiple DNA binding proteins Each nucleotide in the consensus sequence is the nucleotide most often observed at that position in actual sequences TATA box 10 bp upstream from transcription start site Positions of the promoter sequences determine where the RNA polymerase begins synthesis Most differences in promoter strength result from variation in 35 and 10 promoter spacing Generally the closer the promoters are to the consensus sequence the stronger the promoter Mutations that change the nucleotide sequence in a promoter can alter the promoter s strength All promoters require accessory proteins to activate transcription by RNA polymerase in bacteria these consist of sigma factors protein allows RNA polymerase to bind to promoter Proper binding of Pol II to the promoter also requires a set of 26 general transcription factors Sequence motifs such as enhancers and silencers bind proteins that interact with the transcriptional machinery to regulate the level of transcription RNA polymerase is a molecular machine for transcription Access to naked promoter DNA occurs by transient displacement of the nucleosomes through the action of chromatin remodeling complexes ONE Chain initiation TATA box binding protein binds the promoter DNA and bends it almost a 90 degree angle TWO Chain elongation transcription bubble consists of 15 unwound DNA nucleotides 8 9 of which is paired with the 3 RNA THREE Chain termination RNA polymerase reaches transcription termination sequence in DNA either self terminating or req protein Bacteria typically undergo self termination The shortest RNA molecules are at promoter end and longest are at the gene terminus A class of Lac mutations denoted p p mutations were closely linked to the lacZ gene and any p mutation eliminated activity of a wildtype lacZ gene present in the same DNA molecule Messenger RNA directs the synthesis of a polypeptide chain The RNA molecule produced from a DNA template is the primary transcript Each gene has only one DNA strand that serves as the template strand In prokaryotes the primary transcript serves directly as the mRNA used in polypeptide synthesis Initiation of polypeptide synthesis may begin many nucleotides downstream from the 5 end The untranslated 5 segment of RNA is called the 5 untranslated region which is followed by an open reading frame ORF which specifies the polypeptide chain 500 3000 bp The 3 end of an mRNA molecule following the ORF also is not translated called the 3 untranslated region The short lifetime of prokaryotic mRNA is an important factor in regulating gene activity 8 4 RNA processing converts the original RNA transcript into messenger RNA The primary transcript in eukaryotes must be converted into mRNA called RNA processing The 5 end is altered by addition of modified guanosine in 5 5 linkage terminal group called the cap necessary for ribosome to bind with mRNA 3 end adds poly A tail 200 A bearing nucleotides regulates mRNA stability Introns removed by splicing rejoining exons coding sequences is known as RNA splicing The events that constitute RNA processing begin even while transcription is still in progress and the events are coupled processes which means that occurrence of one event initiates the next Splicing removes introns from the RNA transcript spliceosomes Splicesomes are composed of protein and several small nuclear ribonuceloprotein particles snRNPs The ends of the intron are brought together by U1 RNA which forms base pairs with nucleotides in the intron at both the 5 and 3 ends U2 RNA binds to the branchpoint region and interacts with paired U4 U6 RNAs ends up pairing with U6 and intron of transcript U5 RNA helps line up the two exons and