LECTURE 3 Gene Transcription and RNA Modification (Chapter 12)

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

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

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

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

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

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

Is this simplistic?

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?

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

Transcription Figure 12.3 DNA of a gene Promoter Terminator Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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

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

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

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!

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

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

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 12.4 The conventional numbering system of promoters

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 12.4 The conventional numbering system of promoters

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

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

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 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. HELIX Figure 12.6

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

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

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