Biol115 The Thread of Life - Sham...

Biol115The Thread of Life

Lecture 6

Getting the message:

DNA transcription

“Understanding how DNA transmits all it knows about cancer, physics, dreaming

and love will keep man searching for some time”

~David R. Brower

Principles of Biology

• Chapter ‘Gene Expression’ (The expression of genes)

• Chapter ‘Transcription’

Biol115_2014_Lecture 6 2

Objectives

• Explain the processes that occur during the three phases of transcription.

• Describe the molecular factors that aid in transcription.

• Relate the importance of specific sequences on the DNA molecule to the process of transcription.

• Describe the differences between eukaryotic and prokaryotic transcription.

• Describe RNA processing.

• Key terms: elongation, initiation, intron, promoter, RNA polymerase, TATA box, termination, transcription, transcription initiation complex, transcription start site


Gene expression: the cookbook analogy

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mRNA

polypeptide/protein

translationtranscription

DNA

Basic principles of transcription

• Transcription is the synthesis of RNA under the direction of DNA

• Transcription is mediated by the enzyme, RNA polymerase

• Transcription produces messenger RNA (mRNA)

RNA polymerase and

transcription


• In prokaryotes, mRNA produced by transcription is immediately translated without more processing

• In a eukaryotic cell, the nuclear envelope separates transcription from translation

• Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

Basic principles of transcription


Synthesis of an RNA transcript

The three stages of transcription:

• Initiation

• Elongation

• Termination


RNA polymerase binding and initiation of transcription

• Promoters signal the initiation of RNA synthesis

• Transcription factors mediate the binding of RNA polymerase

and the initiation of transcription

• The completed assembly of transcription factors and RNA

polymerase II bound to a promoter is called a transcription

initiation complex

• A promoter called a TATA box is crucial in forming the initiation

complex in eukaryotes


Elongation of the RNA strand

• As RNA polymerase moves along the DNA, it untwists the double

helix, 10 to 20 bases at a time

• Transcription progresses at a rate of 40 nucleotides per second in

eukaryotes

• A gene can be transcribed simultaneously by several RNA

polymerases


Termination of transcription

• The mechanisms of termination are different in bacteria and

eukaryotes

• In bacteria, the polymerase stops transcription at the end of the

terminator

• In eukaryotes, the polymerase continues transcription after the

pre-mRNA is cleaved from the growing RNA chain; the

polymerase eventually falls off the DNA


Transcription in prokaryotes


Multiple RNA polymerases

transcribing the same gene

simultaneously!

Nature 417, 967-970.

RNA polymerase binding and initiation of transcription

• In eukaryotes, transcription factors

mediate the binding of RNA polymerase

and the initiation of transcription

• A sequence in the upstream regulatory

region, called a TATA box, is crucial in

forming the initiation complex in eukaryotes


The eukaryotic promoter

Promoters are the start sites of transcription. Eukaryotic promoters can include the TFIIB

recognition element, the TATA box, the initiator element and the downstream promoter element. Not

all of these are present in every promoter.


Termination of transcriptionRNA polymerase stops transcribing at the end of a gene – ‘stop’

defined by termination signals (example shown)

Termination signals (Gould and Keeton, Biological Sciences)

Biol115_2014_Lecture 6

Hairpin loops and poly-U tails (Gould and Keeton, Biological Sciences)

14

Eukaryotic cells modify RNA after transcription

• Enzymes in the eukaryotic nucleus modify pre-mRNA before

the genetic messages are dispatched to the cytoplasm

• During RNA processing, both ends of the primary transcript

are usually altered

• Also, usually some interior parts of the molecule are cut out,

and the other parts spliced together


Alteration of mRNA ends

• Each end of a pre-mRNA molecule is modified in a particular

way:

• The 5 end receives a modified nucleotide 5 cap

• The 3 end gets a poly-A tail

• These modifications share several functions:

• They seem to facilitate the export of mRNA

• They protect mRNA from hydrolytic enzymes

• They help ribosomes attach to the 5 end (for translation)


Insert Fig. 17-9 P336


Split genes and RNA splicing• Most eukaryotic genes and their RNA transcripts have long noncoding stretches

of nucleotides that lie between coding regions

• These noncoding regions are called intervening sequences, or introns

• The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences

• RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

• Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognise the splice sites


Example of mRNA splicing

DNAASDFASISASDFTHECGHDTHREADARETVTOFSRVTLIFE

DNAISTHETHREADOFLIFE


E1 I1 E2 I2 E3 I3 E4 I4 E5 I5 E6

pre-mRNA

mature mRNA

E = exon

I = intron

19

Insert Fig. 17-10 P337


RNA splicing

In eukaryotes, coding regions,

or exons, are interspersed

with non-coding introns.

Introns are removed, and

exons are joined together

during RNA splicing to

produce a mature mRNA

molecule.


mRNA for the egg protein, ovalbumin, being spliced to

remove introns


Gene Pre-mRNA mRNA

Factor 8 200,000 nt 10,000 nt

Duchenne

muscular

dystrophy

2,000,000 nt 16,000 nt

22

• Proteins often have a modular architecture consisting of discrete regions called domains

• In many cases, different exons code for the different domains in a protein

• Exon shuffling may result in the evolution of new proteins

The functional and evolutionary importance of introns


The functional and evolutionary importance of introns

• Some genes can encode more than one kind of polypeptide,

depending on which segments are treated as exons during

RNA splicing

• Such variations are called alternative RNA splicing

• Because of alternative splicing, the number of different

proteins an organism can produce is much greater than its

number of genes


Alternative splicing: expressing several proteins from a single gene.


Even though it is

translated from a

single gene, the

information within

an mRNA

transcript can be

rearranged during

mRNA splicing.

The final mRNA

would be

translated into a

protein with a

different number or

order of protein

subunits than what

was coded for by

the pre-spliced

mRNA.

The Drosophila Dscam gene, involved in

adhesion between neurons, contains 4

clusters of exons, each with array of

possible exons. These are spliced into the

mRNA in an exclusive fashion, so that

only one of each of the possible exons is

represented. If all combinations of these

exons are used in alternative splicing, the

Dscam gene can produce 38,016 different

proteins.

You should now be able to:

1. Briefly explain how information flows from gene to protein.

2. Compare transcription in bacteria and eukaryotes.

3. Include the following terms in a description of transcription: mRNA,

RNA polymerase, the promoter, the terminator, the transcription unit,

initiation, elongation, termination, splicing and introns.

4. Explain three types of post-transcriptional processing of eukaryotic

pre-mRNA.

5. Suggest reasons for the occurrence of introns in eukaryotic genes and

the possible evolutionary benefits that they confer.


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