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Central dogma of Genetics • A Gene is a DNA sequence • A Gene encodes for a protein • A protein could function as an enzyme or a biocatalyst that is responsible for allowing chemical reactions to take place in a cell. • Many chemical reactions together produce a trait or characteristic like flower color. Genes to Traits • Genes ----->proteins -----> Traits (Red flower color) The Information Within the DNA Is Accessed During the Process of Gene Expression • Gene expression occurs in two steps – Transcription • The genetic information in DNA is copied into a nucleotide sequence of ribonucleic acid (RNA) – Translation • The nucleotide sequence in RNA is converted (using the genetic code) into the amino acid sequence of a protein Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display TRANSCRIPTION • Transcription literally means the act or process of making a copy • In genetics, the term refer to the copying of a DNA sequence into an RNA sequence • The structure of DNA is not altered as a result of this process – It can continue to store information Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Gene Expression Structural genes encode the amino acids of a polypeptide Transcription of a structural gene produces messenger RNA, usually called mRNA Polypeptide synthesis is called translation The mRNA sequence determines the amino acids in the polypeptide The function of the protein determines traits This path from gene to trait is called the central dogma of genetics Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The central dogma of genetics Signals the end of protein synthesis Gene Expression Requires Base Sequences The strand that is actually transcribed (used as the template) is termed the template strand The opposite strand is called the coding strand or the sense strand as well as the nontemplate strand The base sequence is identical to the RNA transcript Except for the substitution of uracil in RNA for thymine in DNA Transcription factors recognize the promoter and regulatory sequences to control transcription mRNA sequences such as the ribosomal-binding site and codons direct translation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Stages of Transcription Transcription occurs in three stages Initiation Elongation Termination These steps involve protein-DNA interactions Proteins such as RNA polymerase interact with DNA sequences Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transcription Most of the promoter region is labeled with negative numbers Bases preceding this are numbered in a negative direction There is no base numbered 0 Bases to the right are numbered in a positive direction The conventional numbering system of promoters Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Sequence elements that play a key role in transcription The promoter may span a large region, but specific short sequence elements are particularly critical for promoter recognition and activity level Sometimes termed the Pribnow box, after its discoverer \The conventional numbering system of promoters Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display For many bacterial genes, there is a good correlation between the rate of RNA transcription and the degree of agreement with the consensus sequences The most commonly occurring bases Examples of –35 and –10 sequences within a variety of bacterial promoters Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Initiation of Bacterial Transcription RNA polymerase is the enzyme that catalyzes the synthesis of RNA In E. coli, the RNA polymerase holoenzyme is composed of Core enzyme Sigma factor Five subunits = a2bb’ One subunit = s These subunits play distinct functional roles Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Binding of factor protein to DNA double helix Amino acids within the a helices hydrogen bond with bases in the promoter sequence elements Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Similar to the synthesis of DNA via DNA polymerase rho utilization site Rho protein is a helicase r-dependent termination r-dependent termination • r-independent termination is facilitated by two sequences in the RNA – 1. A uracil-rich sequence located at the 3’ end of the RNA – 2. A stem-loop structure upstream of the Us URNA-ADNA hydrogen bonds are very weak This type is also called intrinsic r-independent termination Stabilizes the RNA pol pausing Eukaryotic RNA Polymerases Nuclear DNA is transcribed by three different RNA polymerases RNA pol I Transcribes all rRNA genes (except for the 5S rRNA) RNA pol II Transcribes all structural genes Thus, synthesizes all mRNAs Transcribes some snRNA genes RNA pol III Transcribes all tRNA genes And the 5S rRNA gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Eukaryotic RNA Polymerases All three are very similar structurally and are composed of many subunits There is also a remarkable similarity between the bacterial RNA pol and its eukaryotic counterparts Structure of RNA polymerase © From Patrick Cramer, David A. Bushnell, Roger D. Kornberg. "Structural Basis of Transcription: RNA Polymerase II at 2.8 Ångstrom Resolution." Science, Vol. © From Seth Darst, Bacterial RNA polymerase. 292:5523, 1863-1876, June 8, 2001. Current Opinion in Structural Biology. Reprinted with permission of the author. (a) Structure of a bacterial RNA polymerase 5′ Structure of a eukaryotic RNA polymerase II (yeast) 3′ Transcribed DNA (upstream) Lid Exit Clamp Rudder 5′ Entering DNA (downstream) 3′ Wall 5′ Mg2+ Bridge Jaw Catalytic site Transcription NTPs enter through a pore (b) Schematic structure of RNA polymerase Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Usually an adenine • The core promoter is relatively short – It consists of the TATA box • Important in determining the precise start point for transcription • The core promoter by itself produces a low level of transcription – This is termed basal transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Usually an adenine • The core promoter is relatively short – It consists of the TATA box • Important in determining the precise start point for transcription • The core promoter by itself produces a low level of transcription – This is termed basal transcription Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display • Regulatory elements affect the binding of RNA polymerase to the promoter – They are of two types • Enhancers – Stimulate transcription • Silencers – Inhibit transcription – They vary widely in their locations but are often found in the –50 to –100 region Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A closed complex RNA pol II can now proceed to the elongation stage Released after the open complex is formed Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transcription termination RNA polymerase II • RNA polymerase II transcribes a gene past the polyA signal sequence. • The RNA is cleaved just past the polyA signal sequence. RNA polymerase continues transcribing the DNA. Possible mechanisms for Pol II termination RNA polymerase II transcribes a gene past the polyA signal sequence. 5′ 3′ PolyA signal sequence The RNA is cleaved just past the polyA signal sequence. RNA polymerase continues transcribing the DNA. 5′ 3′ 3′ 3′ 5′ 3′ 5′ 3′ 5′ 3′ Exonuclease catches Exonuclease up to RNA polymerase II and causes termination. 5′ 3′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 3′ RNA MODIFICATION • In eukaryotes, the genes are interrupted: the coding sequences, called exons, are interrupted by intervening sequences or introns • Transcription produces the entire gene product – Introns are later removed or excised – Exons are connected together or spliced • This phenomenon is termed RNA splicing – It is a common genetic phenomenon in eukaryotes – Occurs occasionally in bacteria as well Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA MODIFICATION • Aside from splicing, RNA transcripts can be modified in several ways – For example • Trimming of rRNA and tRNA transcripts • 5’ Capping and 3’ polyA tailing of mRNA transcripts Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display This processing occurs in the nucleolus Functional RNAs that are key in ribosome structure Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Found to contain both RNA and protein subunits Therefore, it is a ribozyme Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Covalently modified bases Pre-mRNA Splicing The spliceosome is a large complex that splices pre-mRNA It is composed of several subunits known as snRNPs (pronounced “snurps”) Each snRNP contains small nuclear RNA and a set of proteins Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Intron RNA is defined by particular sequences within the intron and at the intro-exon boundaries The consensus sequences for the splicing of mammalian pre-mRNA are shown here Sequences shown in bold are highly conserved Corresponds to the boxed adenine Serve as recognition sites for the binding of the spliceosome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Intron loops out and exons brought closer together Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Cleavage may be catalyzed by RNA molecules within U2 and U6 Intron will be degraded and the snRNPs used again Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Capping of pre-mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA editing Change in the nucleotide sequence of an RNA Can involve addition or deletion of particular bases Can also occur through conversion of a base First discovered in trypanosomes Now known to occur in many organisms Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA editing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display