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 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 TRANSCRIPTION

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

The central dogma of genetics

Signals the end of protein synthesis

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 Gene Expression Requires Base Sequences

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transcription occurs in three stages Initiation Elongation Termination These steps involve protein-DNA interactions Proteins such as RNA polymerase interact with DNA sequences The Stages of Transcription

Transcription

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The conventional numbering system of promoters 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 Most of the promoter region is labeled with negative numbers

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display \ The conventional numbering system of promoters 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 Sequence elements that play a key role in transcription

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Examples of –35 and –10 sequences within a variety of bacterial promoters 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

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 =  2 bb ’  Sigma factor One subunit =  These subunits play distinct functional roles Initiation of Bacterial Transcription

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Amino acids within the a helices hydrogen bond with bases in the promoter sequence elements Binding of  factor protein to DNA double helix 

Similar to the synthesis of DNA via DNA polymerase

rho utilization site  -dependent termination Rho protein is a helicase

 -dependent termination

 -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  -independent termination U RNA -A DNA hydrogen bonds are very weak This type is also called intrinsic Stabilizes the RNA pol pausing

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

© From Seth Darst, Bacterial RNA polymerase. Current Opinion in Structural Biology. Reprinted with permission of the author. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 Eukaryotic RNA Polymerases Structure of RNA polymerase (a) Structure of a bacterial RNA polymerase (b) Schematic structure of RNA polymerase Structure of a eukaryotic RNA polymerase II (yeast) Transcribed DNA (upstream) 5′ Jaw Clamp Rudder Catalytic site Wall Bridge NTPs enter through a pore Mg 2+ Entering DNA (downstream) Transcription Lid Exit 3′ 5′ © 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, , June 8, 2001.

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

A closed complex Rele ased after the open complex is formed RNA pol II can now proceed to the elongation stage

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. The RNA is cleaved just past the polyA signal sequence. RNA polymerase continues transcribing the DNA. PolyA signal sequence 3′ 5′ 3′ 5′ 3′ 5′ Exonuclease catches up to RNA polymerase II and causes termination. Exonuclease 3′ 5′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

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 RNA MODIFICATION

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Functional RNAs that are key in ribosome structure This processing occurs in the nucleolus

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Foun d to contain both RNA and protein subunits Therefore, it is a ribozyme Covalently modified bases

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 Pre-mRNA Splicing

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

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

Capping of pre-mRNA

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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 RNA editing

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