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 ----->proteins -----> Traits (Red flower color) Genes to Traits

Gene expression occurs in two steps 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 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

Structural genes encode the amino acids of a polypeptide 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

The conventional numbering system of promoters 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 Sequence elements that play a key role in transcription 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

The most commonly occurring bases 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 Five subunits = a2bb’ Sigma factor 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 protein is a helicase 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 Stabilizes the RNA pol pausing URNA-ADNA hydrogen bonds are very weak This type is also called intrinsic r-independent termination

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. 292:5523, 1863-1876, June 8, 2001. © From Seth Darst, Bacterial RNA polymerase. Current Opinion in Structural Biology. Reprinted with permission of the author. (a) Structure of a bacterial RNA polymerase Structure of a eukaryotic RNA polymerase II (yeast) 5′ 3′ Transcribed DNA (upstream) Lid Exit Clamp 5′ Rudder Entering DNA (downstream) Wall 3′ 5′ Mg2+ Bridge Jaw Catalytic site through a pore NTPs enter Transcription (b) Schematic structure of RNA polymerase Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The core promoter is relatively short It consists of the TATA box 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

The core promoter is relatively short It consists of the TATA box 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

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Released after the open complex is formed 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.

Transcription produces the entire gene product 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

Aside from splicing, RNA transcripts can be modified in several ways 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 Covalently modified bases Therefore, it is a ribozyme Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The spliceosome is a large complex that splices pre-mRNA 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 Corresponds to the boxed adenine Sequences shown in bold are highly conserved 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

Intron will be degraded and the snRNPs used again 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

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RNA editing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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′ Allosteric model: After passing the polyA signal sequence, RNA polymerase II is destabilized due to the release of elongation factors or the binding of termination factors (not shown). Termination occurs. Torpedo model: An exonuclease binds to the 5′ end of the RNA that is still being transcribed and degrades it in a 5′ to 3′ direction. 5′ 5′ 3′ 3′ 5′ 3′ Exonuclease catches up to RNA polymerase II and causes termination. Exonuclease 3′ 5′ 3′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display