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CHAPTER 11. Gene Expression: From Transcription to Translation. 11.1 The Relationship between Genes and Proteins (1). Genes store information for producing all cellular proteins. Early observation suggested a direct relationship between genes and proteins.
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CHAPTER 11 Gene Expression: From Transcription to Translation
11.1 The Relationship between Genes and Proteins (1) • Genes store information for producing all cellular proteins. • Early observation suggested a direct relationship between genes and proteins. • Garrod studied the relationship between a specific gene, a specific enzyme, and a metabolic condition (alcaptonuria). • Beadle and Tatum formulated the “one gene–one enzyme” hypothesis.
The Relationship between Genes and Proteins (2) • Beadle and Tatum’s hypothesis was alter modified to “one gene–one polypeptide chain” • Mutation in a single gene causes a single substitution in an amino acid sequence of a single protein.
The Relationship between Genes and Proteins (3) • An Overview of the Flow of Information through the Cell • Messenger RNA (mRNA) is an intermediate between a gene and a polypeptide. • Transcription is the process by which RNA is formed from a DNA template. • Translation is the process by which proteins are synthesized in the cytoplasm from an mRNA template.
The Relationship between Genes and Proteins (4) • There are three classes of RNA in a cell: mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA). • rRNA recognizes other molecules, provide structural support, and catalyzes the chemical reaction in which amino acids are linked to one another. • tRNAs are required to translate information in the mRNA code into amino acids.
11.2 An Overview of Transcription andTranslation in Both Prokaryoticand Eukaryotic Cells (1) • DNA-dependent RNA polymerases (or RNA polymerases) are responsible for transcription in both prokaryotes and eukaryotes. • These enzymes incorporate nucleotides into a strand of RNA from a DNA template. • The promoter is where the enzyme binds prior to initiating transcription. • The enzyme require the help of transcription factors to recognize the promoter.
An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (2) • The newly synthesized RNA chain grows in a 5’ to 3’ direction antiparallel to the DNA. • RNA polymerase must be processive – remain attached to DNA over long stretches. • RNA polymerase must be able to move from nucleotide to nucleotide. • Nucleotides enter the polymerization reaction as trinucleotide precursors. • The reaction is driven forward by the hydrolysis of a pyrophosphate: PPi 2Pi
Experimental techniques to follow the activities of RNA polymerase
An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (3) • Once polymerase has finished adding nucleotides, the DNA-RNA hybrid dissociates and the DNA double helix reforms. • There are two enzymatic activities of RNA polymerase: digestion of incorrect nucleotides and polymerization.
An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (4) • Transcription in Bacteria • There is only one type of RNA polymerase in prokaryotes: five subunits associated to form a core enzyme. • Transcription-competent cells also have a sigma factor attached to the RNA polymerase before attaching to DNA.
An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (5) • Bacterial promoters are located upstream from the site of initiation. • Two conserved regions: –35 element (consensus sequence) and Pribnow box. • Differences in the DNA sequences at both –35 element and the Pribnow box may regulate gene expression. • Termination in bacteria can either require a rho factor protein or may reach a terminator sequence without rho.
An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (6) • Transcription and Processing in Eukaryotic Cells • There are three types of RNA polymerases in eukaryotes. • Most rRNAs are transcribed by RNA polymerase I. • mRNAs are transcribed by RNA polymerase II. • tRNAs are transcribed by RNA polymerase III.
A comparison of prokaryotic and eukaryotic RNA polymerase structure
An Overview of Transcription and Translation in Both Prokaryotic and Eukaryotic Cells (7) • Transcription factors regulate the activity of RNA polymerases. • Newly transcribed RNAs are processed. • A primary transcript (or pre-RNA) is the initial RNA molecule synthesized. • A transcription unit is the DNA segment corresponding to a primary transcript. • A variety of small RNAs are required for RNA processing.
11.3 Synthesis and Processing of Ribosomal and Transfer RNAs (1) • A eukaryotic cell may contain millions of ribosomes. • The DNA sequence encoding rRNA are called rDNA, and is typically clustered in the genome. • In nondividing cells, rDNA are clustered in the nucleoli, where ribosomes are produced.
Synthesis and Processing of Ribosomal and Transfer RNAs (2) • Synthesizing the rRNA Precursor • rRNA genes are arranged in tandem. • rRNA transcription has a “Christmas tree” pattern. • Proteins that convert rRNA precursors into mature rRNA become associated with pre-rRNA during transcription. • The nonstranscribed spacer separates transcription units in a ribosomal gene cluster.
Synthesis and Processing of Ribosomal and Transfer RNAs (3) • Processing of the rRNA Precursor • A single primary transcript (pre-rRNA) can be spliced into three rRNAs: 28S, 18S, and 5.8S. • Pre-rRNA contains large numbers of methylated nucleotides and pseudouridine residues. • Unaltered sections of the pre-rRNA are discarded.
Synthesis and Processing of Ribosomal and Transfer RNAs (4) • The Role of snoRNAs • Processing of pre-rRNA is helped by small, nucleolar RNAs (snoRNAs). • snoRNAs are packaged with proteins into snoRNPs (small, nucleolar ribonucleoproteins). • snoRNAs modify bases in pre-RNAs.
Synthesis and Processing of Ribosomal and Transfer RNAs (5) • Synthesis and Processing of the 5S rRNA • The 5S rRNA genes are located outside the nucleolus. • It is transcribed by RNS polymerase III, which uses an internal promoter.
Synthesis and Processing of Ribosomal and Transfer RNAs (6) • Transfer RNAs • tRNA genes are located in small clusters scattered around the genome. • tRNAs have promoter sequences within the coding region of the gene. • During processing, the tRNA precursor is trimmed and numerous bases must be modified.
11.4 Synthesis and Processing of Messenger RNAs (1) • The precursors of mRNAs are represented by diverse RNAs called heterogeneous nuclearRNAs (hnRNAs). • hnRNAS are found only in the nucleus. • hnRNAs have large molecular weights. • hnRNAs are degraded after a very short time.
Synthesis and Processing of Messenger RNAs (2) • The Machinery for mRNA Transcription • RNA polymerase II is assisted by general transcription factors (GTFs) to form the preinitiation complex (PIC). • The critical portion of the promoter lies 24-32 bases upstream from the initiation site, and contains the TATA box. • The preinitiation complex of GTFs and polymerase assemble at the TATA box.
Initiation of transcription from a eukaryotic polymerase II promoter
Initiation of transcription from a eukaryotic polymerase II promoter
Synthesis and Processing of Messenger RNAs (3) • The preinitiation complex assembly starts with the binding of the TATA-binding protein (TBP) to the promoter. • TBP is a subunit of the TFIID and when it binds to the promoter causes a conformation change in DNA.
Structural models of the formation of the preinitiation complex
Synthesis and Processing of Messenger RNAs (4) • Binding of TFIID sets the stage for the assembly of the complete PIC. • The three GTFs bound to the promoter allows the binding of RNA polymerase with its TFIIF. • As long as TFIID remains bound to the promoter, additional RNA polymerases may be able to attach for additional rounds of transcription.
Synthesis and Processing of Messenger RNAs (5) • RNA polymerase is heavily phosphorylated at the carboxyl-terminal domain (CTD). • CTD phosphorylation can be catalyzed by different protein kinases. • TFIIH acts as the protein kinase. • Termination of transcription is not well understood.
Synthesis and Processing of Messenger RNAs (6) • The Structure of mRNAs: Messenger RNAs share certain properties • They each code for a specific polypeptide. • They are found in the cytoplasm. • They are attached to ribosomes when translated. • Most have a noncoding segment. • Eukaryotic mRNAs modifications at their 5’ (guanosone cap) and a 3’ poly(A) tail.
Synthesis and Processing of Messenger RNAs (7) • Split Genes: An Unexpected Finding • The difference between hnRNA and mRNA provided early clues about RNA processing. • Eukaryotic genes contain intervening sequences which are missing from mature mRNAs. • The presence of genes with intervening sequences are called split genes.