1. LECTURE 3 Gene Transcription and RNA Modification (Chapter 12)

2. INTRODUCTION The term gene has many definitions
For this class, a gene is a segment of DNA used to make a product that plays a functional role in the cell
either an RNA or a polypeptide
Transcription is the first step in gene expression

3. Court reporter transcribing court testimony
Words to Know
Transcription: (Verb) The act or process of making a copy
Example: Court reporter hears the witness speaking in English and types a written copy, in English, of the witness’ statements.
Translation: Express the meaning of words or text in another language
Dogma: A principle or set of principles laid down by an authority as incontrovertibly true
Court reporter transcribing court testimony

4. TRANSCRIPTION
In genetics, the term refers to the copying of a DNA sequence into an RNA sequence
Only one strand is copied
The structure of DNA is not altered as a result of this process
It continues to store information and can be transcribed again and again and again

5. 1. Check out
2. Make many copies of the same page
3. Return unaltered
4. Distribute and incite a riot!

6. Gene Expression
Structural genes encode the amino acid sequence of a polypeptide
Transcription of a structural gene produces messenger RNA, usually called mRNA
The mRNA nucleotide sequence determines the amino acid sequence of a polypeptide during translation
The synthesis of functional proteins determines an organisms traits
This path from gene to trait is called the central dogma of genetics
Refer to Figure 12.1

7. The central dogma of genetics
DNA replication:
makes DNA copies that are transmitted
from cell to cell and from parent to
offspring.
Gene
Chromosomal DNA: stores information in
units called genes.
Transcription:
produces an RNA copy of a gene.
Messenger RNA: a temporary copy of a gene
that contains information to
make a polypeptide.
Translation:
produces a polypeptide using the
information in mRNA.
Polypeptide: becomes part of a functional protein
that contributes to an organism's traits.
Figure 12.1

8. Is this simplistic?

9. 12.1 OVERVIEW OF TRANSCRIPTION
Gene expression is the overall process by which the information within a gene is used to produce a functional product which can, in concert with environmental factors, determine a trait
Or: How does a book result in a riot?

10. 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

11. Transcription Figure 12.3 DNA of a gene Promoter Terminator
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DNA of a gene
Transcription
Promoter
Terminator
Initiation: The promoter functions as a recognition
site for transcription factors (not shown). The transcription
factor(s) enables RNA polymerase to bind to the promoter.
Following binding, the DNA is denatured into a bubble
known as the open complex.
5′ end of growing
RNA transcript
Open complex
RNA polymerase
Elongation/synthesis of the RNA transcript:
RNA polymerase slides along the DNA
in an open complex to synthesize RNA.
Termination: A terminator is reached that causes RNA
polymerase and the RNA transcript to dissociate from
the DNA.
Completed RNA
transcript
RNA
polymerase
Figure 12.3

12. RNA Transcripts Have Different Functions
Once they are made, RNA transcripts play different functional roles
Refer to Table 12.1
Well over 90% of all genes are structural genes which are transcribed into mRNA
Final functional products are polypeptides
The other RNA molecules in Table 12.1 are never translated
Final functional products are RNA molecules

13. RNA Transcripts Have Different Functions
The RNA transcripts from nonstructural genes are not translated
They do have various important cellular functions
They can still confer traits
In some cases, the RNA transcript becomes part of a complex that contains protein subunits
For example
Ribosomes
Spliceosomes
Signal recognition particles

14. You don’t need to memorize this slide – however, note how many different types of functional RNA molecules exist and how many different types of functions they perform!

15. 12.2 TRANSCRIPTION IN BACTERIA
Our molecular understanding of gene transcription came from studies involving bacteria and bacteriophages
Indeed, much of our knowledge comes from studies of a single bacterium
E. coli, of course
In this section we will examine the three steps of transcription as they occur in bacteria

16. Promoters Promoters are DNA sequences that “promote” gene expression
More precisely, they direct the exact location for the initiation of transcription
Promoters are typically located just upstream of the site where transcription of a gene actually begins
The bases in a promoter sequence are numbered in relation to the transcription start site
Refer to Figure 12.4

17. Figure 12.4 The conventional numbering system of promoters
Bases preceding the start site are numbered in a negative direction
Most of the promoter region is labeled with negative numbers
There is no base numbered 0
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Coding strand
Transcriptional
start site
Promoter region
–35 sequence
16 –18 bp
–10 sequence +1 5′ 3′ T T C T T T G A A A A A A A A G A A A C T T T T T T 3′ 5′
Template strand 5′ 3′
Bases to the right are numbered in a positive direction
RNA A
Transcription
Figure The conventional numbering system of promoters

18. 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
Coding strand
Transcriptional
start site
Promoter region
–35 sequence
16 –18 bp
–10 sequence +1 5′ 3′ T T C T T T G A A A A A A A A G A A A C T T T T T T 3′ 5′
Template strand
Sometimes termed the Pribnow box, after its discoverer 5′ 3′
RNA A
Transcription
Figure The conventional numbering system of promoters

19. 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

20. Initiation of Bacterial Transcription
The RNA polymerase holoenzyme binds loosely to the DNA
It then scans along the DNA, until it encounters a promoter region
When it does, the sigma factor recognizes both the –35 and –10 regions
A region within the sigma factor that contains a helix-turn-helix structure is involved in a tighter binding to the DNA
Refer to Figure 12.6

21. Binding of factor protein to DNA double helix
Amino acids within the a helices hydrogen bond with bases in the -35 and -10 promoter sequences
Turn
α helices
binding to the
major groove
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HELIX
Figure 12.6

22. A short RNA strand is made within the open complex
The binding of the RNA polymerase to the promoter forms the closed complex
Then, the open complex is formed when the TATAAT box in the -10 region is unwound
A short RNA strand is made within the open complex
The sigma factor is released at this point
This marks the end of initiation
The core enzyme now slides down the DNA to synthesize an RNA strand
This is known as the elongation phase

23. Figure 12.7 RNA polymerase Promotor region σ factor –35 –10
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
RNA polymerase
Promotor region
σ factor
–35
–10
After sliding along the DNA, σ
factor recognizes a promoter, and
RNA polymerase holoenzyme
forms a closed complex.
RNA polymerase
holoenzyme
–35
–10
Closed complex
An open complex is formed, and
a short RNA is made.
–35
–10
Open complex
σ factor is released, and the
core enzyme is able to proceed
down the DNA.
RNA polymerase
core enzyme
–35
–10
σ factor
Figure 12.7
RNA transcript

24. In class skit Characters: Character Played By
A shy female college student
A cute dude
A helpful friend
Dr. Ballard