1. 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.

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

3. 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

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

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

6. 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

7. 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 Copyright © 2006 Pearson Prentice Hall, Inc.

8. 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

9. 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

10. 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

11. 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.

12. 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

13. 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.

14. 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.

15. 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.

16. 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.

17. 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)

18. 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).

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

20. 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?

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

22. 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

23. 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.

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

25. 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

26. 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

27. 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.

28. 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.

29. 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.

30. 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.

31. 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.

32. 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 Copyright © 2006 Pearson Prentice Hall, Inc.

33. 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.

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

35. 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.

36. 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.

37. 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.

38. 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.

39. 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.

40. Alternative Genome
Read article on Alternative Genome

41. Simultaneous Transcription & Translation
FIGURE 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: , 1970 by the American Association for the Advancement of Science.

42. FIGURE 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.