Today… Genome 351, 12 April 2013, Lecture 4 mRNA splicing Promoter recognition Transcriptional regulation Mitosis: how the genetic material is partitioned

Today…Genome 351, 12 April 2013, Lecture 4

•mRNA splicing

• Promoter recognition

• Transcriptional regulation

•Mitosis: how the genetic material is partitioned during cell division

Please be sure to turn in your first problem set assignment today, and also pick up the

second problem sethttp://courses.washington.edu/gen351/

In bacteria (most) mRNAs are co-linear with their corresponding genes

+1

Promoter terminator

bacteria:(pre-mRNA)

(processed mRNA)

eukaryotes:

introns

exons

AACUGACGA

AACTGACGA

mRNA

AACGA

gene

Transcription

Translation

introns are removed during transcription in the nucleus

Events involved in RNA processing

Non-coding

Non-coding

Coding sequence Coding sequenceNoncoding

Exon1 Exon2Intron

Non-coding

Non-coding

Continuous stretch of coding sequence AAAAA

Add a string of A’s to the end

Non-coding

Non-coding

Continuous stretch of coding sequence

Intron removedSplice out the intron

Transport to the cytoplasm

Pre-mRNA

Processed-mRNA

Proteins can be modular-Different regions can have distinct functionsand the modules can correspond to exons

Why does transcript splicing occur?

Interrupted structure allows genes to be modular

secretion cell anchor enzyme binding module

introns

exons

introns can also lie in untranslated sequences

untranslated sequences



introns

exons

introns can also lie in untranslated sequences

untranslated sequencesPre-mRNA:



Pre-mRNA:

Processed-mRNA


secretion cell anchor enzyme binding module AAAA

This form stays anchored to the plasma membrane

Three introns removed

polyA tail added


secretion enzyme binding module

Pre-mRNA:

Processed-mRNA

Alternative splicing or:One mRNAs exon is another one’s

intron!

AAAAsecretion enzyme binding module

This form is secreted

Three introns & an exon removed

one alternative form


enzyme binding module

Pre-mRNA:

Processed-mRNA

Alternative splicing or:One mRNAs exon is another one’s

intron!

AAAAenzyme binding module

This form is retained in the cytoplasm

Three introns & two exons removed

another alternative form

Many additional possibilities with alternative splicing

New York Times

ApoE gene

Promoter

exons

cys cys

112 158

cys arg

arg arg

ApoE2

ApoE3

ApoE4

112 158Allele

10-30-fold increased risk of AD

8%

78%

14%

worldwidefrequency

How do RNA polymerases know where to begin transcription and which way to go?

promoter

mRNA

mRNA

promotergene

genegenemRNA

promoter

First worked out in bacteria by:-comparing sequences near the start sites of transcription of many genes-by studying where RNA polymerase likes to bind to DNA

Comparing sequences at the promoter region of many bacterial genes provides clues:

How do RNA polymerases know where to begin transcription and which way to go?

consensussequence: TTGACAT…15-17bp…TATAAT

transcription start site

direction of transcriptio

n

+1-10 region-35 regionare these important

?

only coding (sense) strand is shown; all sequences 5’-3’

Promoter Strength (# of mRNAs

made/time)

Relatedness of promoter to consensus

TTGACAT…15-17bp…TATAAT -35 -10

ACAGTGA…15-17bp…CTGTCA -35 -10

RNA polymerase binds to the consensus sequences in bacterial promotersRNA polymerase binds to the -35 and -10 regions:

+1-10 region-35 region

TATAAT


n

Would you expect RNA polymerase to bind the other way around and transcribe in the reverse direction?

TTGACAT

RNA polymerase

-35 binding part of RNA polymerase


RNA polymerase binds to the consensus sequences in bacterial promotersRNA polymerase binds to the -35 and -10 regions:


TATAAT


n

Would you expect RNA polymerase to bind the other way around and transcribe in the reverse direction?

TTGACAT

RNA polymerase



RNA polymerase binds to the consensus sequences in bacterial promoters


TATAATTTGACAT

RNA polymerase


n

RNA polymerase

TAATATTACAGTT


n

Would you expect RNA polymerase to bind this sequence and initiate transcription?

5’3’

5’ 3’

5’ TTGACAT 3’ 3’ TTGACAT 5’=These are chemically distinct molecules with different 3-D shapes!!

mRNA

mRNAgene

genegenemRNA

How do RNA polymerases know where to begin transcription and which way to go?In bacteria RNA polymerase binds specific sequences near the start site of transcription that orient the polymerase:

-10 region-35 region

TTGACAT TATAAT

-35 region-10 region

TACAGTTTAATAT

similar principles- but a different mechanism-orients RNA polymerase in eukaryotes

In eukaryotes, RNA polymerase is regulated by DNA-binding proteins

RNA polymerase:

+1RNA polymerase does not efficiently bind to DNA and activate transcription on its own


RNA polymerase:transcription factors (TF’s):

+1RNA polymerase does not efficiently bind to DNA and activate transcription on its own

+1

But TF’s that bind to specific DNA sequences & to RNA polymerase can recruit RNA polymerase & activate transcription


RNA polymerase:

Some TF’s can also inhibit transcription

transcription factors (TF’s):

+1

+1

But TF’s that bind to specific DNA sequences & to RNA polymerase can recruit RNA polymerase & activate transcription

RNA polymerase does not efficiently bind to DNA and activate transcription on its own

Switches and Regulators - A Metaphor

• Switches control transcription (which take the form of DNA sequence)- Called regulatory elements (RE’s) or enhancers- Adjoin the promoter region, but can be quite distant

• Regulators, which take the form of proteins that bind the DNA, operate the switches- Called transcription factors (TF’s)

• When and how much RNA is made often is the product of multiple elements and regulators

Control of gene expression

• Each cell contains the same genetic blueprint

• Cell types differ in their protein content

• Some genes are used in almost all cells (housekeeping genes)

• Other genes are used selectively in different cell types or in response to different conditions.

An imaginary regulatory region

Promoter

RE1 RE2 RE3 RE4 RE5 RE6

Controls timing of transcription

Inhibits transcription

Increases transcription

Turns on in brain

Antennapedia gene is normally only transcribed in the thorax; legs are made.

A mutant promoter causes the Antennapedia gene to be expressed in the thorax and also in the head, where legs result instead of antennae!

Example: Antennapedia gene in fruit flies

Expressing a regulatory gene in the wrong place can have disastrous consequences!!!

Lactose tolerance: A human example of a promoter mutation

Lactase levels in humansLa

ctase

levels

Age in years2 10

lactose intolerant

lactose tolerant

World wide distribution of lactose intolerance

Convergent evolution: independent acquisition of the same biological trait in distinct populations

The cellular life cycle

fertilized egg; a single cell!

How is the genetic material equally divided during

mitosis?

The formation of sperm and eggs-more later on this subject

Mitosis: dividing the content of a

cell

Chromosomes - a reminder

How many do humans have?

Photo: David McDonald, Laboratory of Pathology of Seattle

• 22 pairs of autosomes• 2 sex chromosomes

• Each parent contributes one chromosome to each pair

• Chromosomes of the same pair are called homologs

• Others are called non-homologous

Homologous and non-homologous chromosomes

1p 1m

2p 2m

3p 3m

21p

22m22p

21m

Xp or Y Xm

homologous

non-homologous

The zygote receives one paternal (p)

and one maternal (m) copy of each

homologous chromosome

homologous

The DNA of human chromosomes

# genes# base pairs # genes# base pairs


cell growth; chromosome duplication

chromosomes

decondensed


Elements of mitosis:

What are decondensed chromosomes?

How are chromosomes duplicated?

Chromosome structure – a reminder

chromosome structure during cell growth & chromosome replication (decondensed)

a condensed chromosome

Chromosome replication – a reminder

• Mechanism of DNA synthesis ensure that each double stranded DNA gets copied only once.

• The products of DNA replication have one new DNA strand and one old one (semi-conservative replication)



chromosome segregationcell growth;

chromosome duplication

chromosomes

decondensedchromosome segregation

chromosomes condensed

repeat


only showing a single duplicated homolog – 45 others not shown

Chromosome structure – a reminder

chromosome structure during cell growth & chromosome replication (decondensed)

a condensed chromosome

sister chromatids; double-stranded DNA copies of the SAME homolog

held together at the centromere





chromosomes



repeat


only showing a single duplicated homolog – 45 others not shown





chromosomes



repeat


Mitosis -- making sure each daughter cell gets one copy of each pair of

chromosomes

Understand what’s happening to the chromosomes!

•Copied chromosomes (sister chromatids) stay joined together at the centromere.

•Proteins pull the two sister chromatids to opposite poles

•Each daughter cell gets one copy of each homolog.

Mitosis -- homologous chromosomes

1m 1p

2 copies 1m 2 copies 1p

1m

1p1m

1p

joined at centromere

2 copies 1m

1m

1p1m

1p

2 copies 1p

exact copies

Mitosis – following the fate of CFTR

1m 1p

2 copies 1m 2 copies 1p

1m

1p1m

1p

joined at centromere

2 copies 1m

1m

1p1m

1p

2 copies 1p

exact copies

Mitosis – following the fate of CFTR

CFTR+

CFTR-CFTR+

CFTR-

2 copies CFTR+ 2 copies CFTR-

2 copies CFTR+

2 copies CFTR-

CFTR+

CFTR-CFTR+

CFTR-

exact copies

CFTR+ CFTR-

A CFTR heterozygote (CFTR+/CFTR-)

GTGCACCTGACTCCTGAGGAG

CTCCTCAGGAGTCAGGTGCAC

GTGCACCTGACTCCTGTGGAG

CTCCACAGGAGTCAGGTGCAC

Mitosis -- 2 copies of each chromosome at the start

Paternal chromosome

Maternal chromosome

A closer look at the chromosomes





DNA strands separate followed by new strand synthesis




GTGCACCTGACTCCTGTGGAGCTCCACAGGAGTCAGGTGCAC



• Mitosis -- after replication 4 copies

• Homologs unpaired; sister chromatids joined by centromere





CTCCTCAGGAGTCAGGTGCACGTGCACCTGACTCCTGTGGAG




Each daughter has a copy of each homolog




Mitosis and the cell cycle

DNA synthesis

Chromosome condensation

Chromosome alignment

One copy of each chromosome to each daughter

Nuclear membrane breakdown

Mitosis vs. Meiosis

Meiosis: the formation of gametes

Mitosis: dividing somatic cells

- The goal of mitosis is to make more “somatic” cells:each daughter cell should have the same chromosome set as the parental cell

- The goal of meiosis is to make sperm and eggs:each daughter cell should have half the number of chromosome sets as the parental cell

number of copies of any given chromosome/cell (n):

number of copies of any given chromosome/sperm or egg:

2

1

1n

1n

2n 2n

2n 2n

2n 2n

4n!!

2n 2n

1n 1n

2nzygote:

Why reduce the number of chromosome sets during meiosis?

2n = diploid

1n = haploid

Meiosis: the formation of gametes

The challenge:• ensuring that

homologues are partitioned to separate gametes

The solution:• Hold homologous

chromosomes together by crossing over

• target homologues to opposite poles of the cell…

• then separate the homologues3 other combinations

possible!

1n 1n

2n 2n

Documents

Today… Genome 351, 12 April 2013, Lecture 4 mRNA splicing Promoter recognition Transcriptional regulation Mitosis: how the genetic material is partitioned