The Genetic Code and Transcription Active Lecture PowerPoint® Presentation for Essentials of Genetics Seventh Edition Klug, Cummings, Spencer, Palladino Chapter 12 The Genetic Code and Transcription Copyright © 2010 Pearson Education, Inc.

Outline Overview of gene expression How is genetic information encoded? How is information transferred from DNA to RNA Differences between Prokaryotes & Eukaryotes Summary (animation)

Gene Expression Gene Expression DNA mRNA Protein Transcription Translation Step 1 Step 2 Genetic information is stored in DNA. How does a cell use this information to make proteins such as hemoglobin, muscle proteins and enzymes that catalyze biological reactions. This is a two step process: First the nucleotide sequence in DNA is transcribed to RNA. Then the information in RNA is used in making proteins. Gene Expression

Gene Expression Step 2 Translation Step 1 Transcription FIGURE 12-1 Flowchart illustrating how genetic information encoded in DNA produces protein. Step 2 Translation

Gene Expression How is genetic information encoded? The Genetic code How does the information transferred from DNA to RNA? Transcription

The Genetic Code Written in linear form Uses ribonucleotide bases that compose mRNA molecules as “letters” Sequence of RNA is derived from the complementary bases in the template strand of DNA

Figure 13-7 Copyright © 2006 Pearson Prentice Hall, Inc. The Genetic Code The genetic code shows which amino acid to make from the various codons possible in mRNA; It is really important that you understand how to read the genetic code properly. There are 64 possible codons, but only 20 amino acids in proteins. Therefore more than one codon can specify one amino acid. The Genetic code is nearly universal – used by all living organisms; Figure 13-7 Copyright © 2006 Pearson Prentice Hall, Inc.

The Genetic Code In mRNA, triplet codons specify one amino acid Code contains “start” and “stop” signals Code is unambiguous, degenerate, commaless, nonoverlapping, and nearly universal

The Genetic Code The initial amino acid incorporated into all proteins is methionine or a modified form of methionine (fmet) AUG is the only codon to encode for methionine When AUG appears internally in mRNA, an unformylated methionine is inserted into the protein

The Genetic Code The degenerate code: 64 codons to specify the 20 amino acids The triplet nature of the code was revealed by frameshift mutations

DNA Problem 1: Following is a sequence of a nontemplate strand of DNA 5’ ATGCGAATTAGTCCGCAT 3’ Assuming that transcription begins with the first nucleotide and ends with the last, write the sequence of the transcript (mRNA) in the conventional form Work out the template strand first. Then do the mRNA.

DNA Problem 2: Using the genetic code, translate the transcript (mRNA sequence) in problem 1 into amino acid sequence nontemplate 5’ ATGCGAATTAGTCCGCAT 3’ template 3’ TACGCTTAATCAGGCGTA 5’ mRNA 5’ AUGCGAAUUAGUCCGCAU 3’ amino acid . . . . . . . . . . . . . . . . . . Amino acid sequence: Met, Arg, Ile, Ser, Pro, His

Effect of Frame-shift mutations FIGURE 12-2 The effect of frameshift mutations on a DNA sequence with the repeating triplet sequence GAG. (a) The insertion of a single nucleotide shifts all subsequent triplet reading frames. (b) The insertion of three nucleotides changes only two triplets, but the frame of reading is then reestablished to the original sequence.

FIGURE 12-2a The effect of frameshift mutations on a DNA sequence with the repeating triplet sequence GAG. (a) The insertion of a single nucleotide shifts all subsequent triplet reading frames. (b) The insertion of three nucleotides changes only two triplets, but the frame of reading is then reestablished to the original sequence.

FIGURE 12-2b The effect of frameshift mutations on a DNA sequence with the repeating triplet sequence GAG. (a) The insertion of a single nucleotide shifts all subsequent triplet reading frames. (b) The insertion of three nucleotides changes only two triplets, but the frame of reading is then reestablished to the original sequence.

Transcription RNA serves as the intermediate molecule between DNA and proteins RNA is synthesized on a DNA template during transcription Transcription selectively copies only certain parts of the genome. Many copies of the transcript of one gene region is made In contrast, replication doubles the entire genome once. At a given time only some of the genes are expressed.

RNA Polymerase Directs RNA Synthesis RNA polymerase directs the synthesis of RNA using a DNA template No primer is required for initiation. RNA polymerase can initiate transcription de novo RNA polymerase uses ribonucleotides (rATP,rCTP, rGTP & rUTP)

Transcription in E. coli RNA polymerase from E. coli contains the subunits 2a, b, b', and s Transcription begins by RNA polymerase binding to template at the promoter The s subunit is responsible for promoter recognition Promoters are found in the beginning of a gene. * mRNA is not made right from the promoter region. mRNA is made several bases from the promoter sequence. Eg: 10 bases from tata box, etc. (in eukaryotes, transcription begins at the beginning of the first exon).

Transcription in E. coli E. coli promoters have two consensus sequences upstream of transcription initiation site: TATAAT positioned at –10 TTGACA positioned at –35

Prokaryotic Promoters First one at -10 bases upstream of the point of transcription This has the consensus sequence of TATAAT. Actual sequence may differ by one nt. Rich in AT. Why?

Steps in Transcription Initiation Elongation Termination To transcribe a gene, RNA polymerase proceeds thru a series of steps.

Transcription Initaition Transcription begins when RNA Polymerase binds to a region of gene known as a Promoter Elongation Transcription proceeds in 5’ to 3’ direction Termination Transcription stops when it reaches a region in the gene known as Terminator

RNA Polymerase & DNA binding The holoenzyme binds to the DNA. First it binds loosely and scans for a promoter. When the promoter is found it binds loosely to the promoter. This stage is known as the closed promoter complex because DNA remains in ds for. The holoenzyme can melt a section of DNA at the promoter to form an open promoter complex. Sigma stimulates conversion from loosely bound promoter to a tightly bound promoter. Sigma selects promoters that RNA polymerase will bind tightly.

Transcription Initiation The first base usually transcribed is T in DNA A in RNA. (See Fig. 6.9)

Transcription Elongation in E. Coli Once initiation completed with synthesis of first 8–9 nucleotides, sigma (s) dissociates and elongation proceeds with the core enzyme Core enzyme (α2 β β’) elongates RNA chain by moving along the DNA template and adding ribonucleotides at the 3’end by forming phosphodiester bonds RNA synthesis causes temporary DNA strand separation. Unwinds DNA in front and reanneals DNA behind. Tension relieved by Topoisomerases

Transcription Termination in E. coli Transcription is terminated by signals within the DNA sequence at the end of the gene Hairpin formation in RNA destabilizes the DNA/RNA hybrid and releases RNA transcript In some cases, termination depends on the rho () termination factor

FIGURE 12-8 The early stages of transcription in prokaryotes, showing (a) the components of the process; (b) template binding at the -10 site involving the  subunit of RNA polymerase and subsequent initiation of RNA synthesis; and (c) chain elongation, after the  subunit has dissociated from the transcription complex and the enzyme moves along the DNA template.

FIGURE 12-8a The early stages of transcription in prokaryotes, showing (a) the components of the process; (b) template binding at the -10 site involving the  subunit of RNA polymerase and subsequent initiation of RNA synthesis; and (c) chain elongation, after the  subunit has dissociated from the transcription complex and the enzyme moves along the DNA template.

FIGURE 12-8b The early stages of transcription in prokaryotes, showing (a) the components of the process; (b) template binding at the -10 site involving the  subunit of RNA polymerase and subsequent initiation of RNA synthesis; and (c) chain elongation, after the  subunit has dissociated from the transcription complex and the enzyme moves along the DNA template.

FIGURE 12-8c The early stages of transcription in prokaryotes, showing (a) the components of the process; (b) template binding at the -10 site involving the  subunit of RNA polymerase and subsequent initiation of RNA synthesis; and (c) chain elongation, after the  subunit has dissociated from the transcription complex and the enzyme moves along the DNA template.

Transcription in Eukaryotes Occurs in the nucleus Is not coupled to translation Requires chromatin remodeling In prokaryotes, Transcription and translation both takes place in the cytoplasm. Both processes can occur simultaneously.

Table 13-7 Copyright © 2006 Pearson Prentice Hall, Inc. RNA polymerase has little proofreading ability. Error rate in RNA polymerase is 1 in 10, 000 bp, compared to 1 in 1 billion base pairs for DNA polymerase. Table 13-7 Copyright © 2006 Pearson Prentice Hall, Inc.

Eukaryotic Promoters TATA box (-35): a core promoter element; transcription factors bind to them and determines start site of transcription CAAT box (-80): highly conserved DNA sequence found within promoter of many genes; recognized by transcription factors Enhancers can be upstream, within, or downstream of the gene; can modulate transcription from a distance TATA box at -10 in Prokayotes is analogous to the TATA box at -35 in Eukaryotes.

Post-transcriptional Editing of Eukaryotic mRNA Addition of a 5’ cap Addition of 3’ poly A tail Splice out introns

FIGURE 12-9 Posttranscriptional RNA processing in eukaryotes. Heterogeneous nuclear RNA (pre-mRNA) is converted to mRNA, which contains a 5' cap and a 3' poly-A tail. The introns are then spliced out.

FIGURE 12-9 part 1 Posttranscriptional RNA processing in eukaryotes. Heterogeneous nuclear RNA (pre-mRNA) is converted to mRNA, which contains a 5' cap and a 3' poly-A tail. The introns are then spliced out.

FIGURE 12-9 part 2 Posttranscriptional RNA processing in eukaryotes. Heterogeneous nuclear RNA (pre-mRNA) is converted to mRNA, which contains a 5' cap and a 3' poly-A tail. The introns are then spliced out.

Introns in Various Eukaryotic Genes FIGURE 12-11 Intervening sequences in various eukaryotic genes. The numbers indicate the number of nucleotides present in various intron and exon regions.

Alternative Splicing Introns present in pre-mRNAs derived from the same gene can be spiced in more than one way Yields group of mRNAs that, upon translation, results in a series of related proteins Through alternate splicing one gene can give rise to more than one protein.

Alternative Genome Read article on Alternative Genome

Simultaneous Transcription & Translation FIGURE 12-14 part 1 An electron micrograph and interpretive drawing of simultaneous transcription of a gene in E. coli. As each transcript is forming, ribosomes attach, initiating simultaneous translation along each strand. PHOTO: O.L. Miller, Jr., Barbara A. Hamkalo, C.A. Thomas, Jr. Science, 169:392-395, 1970 by the American Association for the Advancement of Science.

FIGURE 12-14 part 2 An electron micrograph and interpretive drawing of simultaneous transcription of a gene in E. coli. As each transcript is forming, ribosomes attach, initiating simultaneous translation along each strand.