Unit 6 Gene expression and regulation

Rosalind Franklin

Woman who generated x-ray images of DNA, she provided Watson and Crick with key data about DNA

Edwin Chargaff

analyzed DNA samples from different species
-He found the following rule held true for all species:
-The amount of adenine equals the amount of thymine
-The amount of cytosine equals the amount of guanine

Bad pairs held are held together by

Hydrogen bonds

Adenine and thymine have

two hydrogen bonds

Cytosine and guanine have

three hydrogen bonds

Backbone of DNA

sugar and phosphate

Center of DNA

nucleotides pairing

DNA stands are

antiparallel

antiparallel

One strand runs 5' to 3', other strand runs in opposite, upside-down direction 3' to 5'
-5' end: free phosphate group
-3' end: free hydroxyl group

DNA is the primary source of

heritable information

RNA is the primary source of

heritable information in some viruses

Plasmids

small circular DNA molecules that replicate separately from the bacterial chromosome

Chromosomal DNA

stores information in units called genes

DNA replication

the process of making a copy of DNA

Models of DNA Replication

conservative, semiconservative, dispersive

Conservative model of DNA replication

-The parental strands direct synthesis of an entirely new double stranded molecule
-The parental strands are fully "conserved"

semi-conservative model of DNA replication

-The two parental strands each make a copy of itself
-After one round of replication the two daughter molecules each have one parental and one new strand

Dispersive model of DNA replication

-The material in the two parental strands is dispersed randomly between the two daughter molecules
-After one round of replication the daughter molecules contain a random mix of parental and new DNA

Meselson-Stahl Experiment

Used isotope of nitrogen to change the weight of DNA N15 & N14, demonstrated that the semi-conservative model is the best description of replication.

Steps of DNA replication

initiation, elongation, termination

origins of replication

Sites where the replication of a DNA molecule begins.

replication fork

A Y-shaped region on a replicating DNA molecule where new strands are growing.

Helicase

An enzyme that untwists the double helix of DNA at the replication forks.

Topoisomerase

will help prevent strain ahead of the replication fork by relaxing supercoiling

Primase

An enzyme that joins RNA nucleotides to make the primer.

DNA polymerase III (DNAP III)

attaches to each primer on the parental strand and moves in the 3' to 5' direction

leading strand

3' 5' requires one primer

lagging strand

5' 3' requires many primers

Okazaki fragments

segments of the lagging strand

DNAP I

replaces RNA nucleotides with DNA nucleotides

DNA ligase

joins the okazaki fragments forming a continuous DNA strand

Telomeres

repeating units of short nucleotide sequences that do not code for genes

gene expression

the process by which DNA directs the synthesis of proteins

gene expression stages

transcription and translation

Transcription

the synthesis of RNA using information from DNA
-Allows for the "message" of the DNA to be transcribed
-Occurs in the nucleus

Translation

the synthesis of a polypeptide using information from RNA
-Occurs at the ribosome
-A nucleotide sequence becomes an amino acid sequence

Types of RNA

messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA)

Messenger RNA (mRNA)

Messenger RNA is synthesized during transcription using a DNA template
-mRNA carries information from the DNA (at the nucleus) to the ribosomes in the cytoplasm

Transfer RNA (tRNA)

Transfer RNA molecules are important in the process of translation
-Each tRNA can carry a specific amino acid
-Can attach to mRNA via their anticodon
-A complementary codon to mRNA
-Allow information to be translated into a peptide sequence

Ribosonal RNA (rRNA)

rRNA helps form ribosomes
-Helps link amino acids together

genetic code

DNA contains the sequence of nucleotides that codes for proteins
-The sequence is read in groups of three called the triplet code
-During transcription, only one DNA strand is being transcribed
-Known as the template strand (also known as the noncoding strand, minus strand, or antisense strand)

Anticodon

group of three bases on a tRNA molecule that are complementary to an mRNA codon

triplet code

A set of three-nucleotide-long words that specify the amino acids for polypeptide chains.

template strand

the strand of DNA that specifies the complementary mRNA molecule

Codons

The three-base sequence of nucleotides in mRNA

redundancy

more than one codon code for each amino acid

reading frame

the codons on the mRNA must be read in the correct groupings during translation to synthesize the correct proteins

Transcription steps

initiation, elongation, termination

initiation of transcription

-Transcription begins when RNA polymerase molecules attach to a promoter region of DNA
-Do not need a primer to attach
-Promoter regions are upstream of the desired gene to transcribe

Initiation in Eukaryotes

Promoter region is called TATA box
-Transcription factors help RNA polymerase bind

Initiation in Prokaryotes

RNA polymerase can bind directly to promoter

elongation (transcription)

RNA polymerase opens the DNA and reads the triplet code of the template strand
-Moves in the 3' to 5' direction
-The mRNA transcript elongates 5' to 3'
-RNA polymerase moves downstream
-Pairs complementary RNA nucleotides
-The growing mRNA strand peels away from the DNA template strand
-DNA double helix then reforms

Termination in prokaryotes

Transcription proceeds through a termination sequence
-Causes a termination signal
-RNA polymerase detaches
-mRNA transcript is released and proceeds to translation
-mRNA does NOT need modifications

termination in eukaryotes

RNA polymerase transcribes a sequence of DNA called the polyadenylation signal sequence
-Codes for a polyadenylation signal (AAUAAA)
-Releases the pre-mRNA from the DNA
-Must undergo modifications before translation

Pre-mRNA modification

-5' cap
-Poly-A tail
-RNA splicing

5' cap

the 5' end of the pre-mRNA receives a modified guanine nucleotide "cap"

poly-A tail

-the 3' end of the pre-mRNA receives 50-250 adenine nucleotides
-Both the 5' cap and the poly-A tail function to:
-Help the mature mRNA leave the nucleus
-Help protect the mRNA from degradation
-Help ribosomes attach to the 5' end of the mRNA when it reaches the cytoplasm

RNA splicing

sections of the pre-mRNA, called introns, are removed and then exons are joined together

Introns

intervening sequence, do not code for amino acids

Exons

expressed sections, code for amino acids

alternative splicing

A single gene can code for more than one kind of polypeptide

mature mRNA

In eukaryotes, transcription produces a long RNA, pre-mRNA, which undergoes certain processing events before it exits the nucleus; mature mRNA is the final functional product.

Translation

the synthesis of a
polypeptide using information from
the mRNA
-Occurs at the ribosome
-A nucleotide sequence becomes
an amino acid sequence
-tRNA is a key player in translating
mRNA to an amino acid sequence

Transfer RNA

has an anticodon region which is complementary and antiparallel to mRNA
-tRNA carries the amino acid that the mRNA codon codes for

Ribosomes

Translation occurs at the ribosome
-Ribosomes have two subunits: small and large
-Prokaryotic and eukaryotic ribosomal subunits
differ in size
-Prokaryotes: small subunit (30s) large subunit
(40s)
-Eukaryotes: small subunit (40s) large subunit
(60s)

ribosome subunits structure

The large subunit has three sites: A, P, and E
-A site: amino acid site
-Holds the next tRNA carrying an amino acid
-P site: polypeptide site
-Holds the tRNA carrying the growing polypeptide
chain
-E site: exit site

Translation 3 steps

initiation, elongation, termination

initiation of translation

Translation begins when the small ribosomal subunit
binds to the mRNA and a charged tRNA binds to the
start codon, AUG, on the mRNA
-The tRNA carries methionine
-Next, the large subunit binds

elongation (translation)

Elongation starts when the next tRNA comes into the A site
-mRNA is moved through the ribosome and its codons are
read
-Each mRNA codon codes for
a specific amino acid
-Codon charts are used to
determine the amino acid

steps of elongation in translation

-Codon recognition: the
appropriate anticodon of the
next tRNA goes to the A-site
-Peptide bond formation:
peptide bonds are formed that
transfer the polypeptide to the
A site tRNA
-Translocation: The tRNA in the
A site moves to the P site, the
tRNA in the P site goes to the
E site. The A site is open for
the next tRNA

termination of translation

Termination occurs when a stop codon in the mRNA
reaches the A site of the ribosome
-Stop codons do not code for amino acids
-The stop codon signals for a release factor
-Hydrolyzes the bond that holds the polypeptide
to the P site
-Polypeptide releases

Retrovirus

Retroviruses, like HIV, are an
exception to the standard flow
of genetic information
-Information flows from
RNA to DNA
-Uses an enzyme
known as reverse
transcriptase
-Couples viral RNA
to DNA
-DNA then becomes
part of the RNA

Gene expression

Prokaryotes and eukaryotes must be able to
regulate which genes are expressed at any given
time
-Genes can be turned "on" or "off" based on
environmental and internal cues
-On/off refers to whether or not transcription will
take place
-Allows for cell specialization

operons

a group of genes that can be turned on
or off

3 parts of operon

promoter, operator, genes

Promoter

where RNA polymerase can attach

operater

the on/off switch

Genes

code for related enzymes in pathway

repressable operon

On to off
-transcription is usually on, but can be repressed (stopped)

inducible operon

Off to on
-transcription is usually
off, but can be induced (started)

regulatory gene

produces a repressor protein that binds to the operator to block RNA polymerase from transcribing the gene
-Always expressed, but at low levels
-Binding of a repressor to an operator is
reversible

differential gene expression

the expression of different genes by cells with the same genome

Eukaryotic gene expression

\-Eukaryotic gene expression is regulated at different stages -Chromatin structure

-Histone acetylation

\-DNA methylation

-Epigenetic Inheritance

-Transcription Initiation 

\-RNA processing

\-Translation Initiation

Chromatin structure

If DNA is tightly wound it is less
accessible for transcription

histone acetylation

adds acetyl groups to histones, which loosens the DNA

DNA methylation

adds methyl groups to DNA, which causes the chromatin to condense

epigenetic inheritance

Chromatin modifications do not alter the nucleotide sequence of the DNA, but they can be heritable to future generations
-Modifications can be reversed, unlike mutations

transcription initiation

Once chromatin modifications allow the DNA to be more accessible, specific transcription factors bind to control elements
-Sections of non coding DNA that serve as binding sites
-Gene expression can be increased or decreased by binding of activators or repressors to control elements

RNA processing

Alternative splicing of pre-mRNA

translation initiation

Translation can be activated or repressed by initiation factors
-MicroRNAs and small interfering RNAs can bind to mRNA and degrade it or block
translation

eukaryotic development

During embryonic development, cell division and cell differentiation occurs
-Cells become specialized in their structure and
function

Morphogenesis

the physical process that gives an organism its shape

cytoplasmic determinants

substances in the maternal egg that influence cells

Induction

cell to cell signals that can cause a change in gene expression

Mutations

changes in the genetic material of a
cell, which can alter phenotypes

Mutation facts

-Primary source of genetic variation
-Normal function and production of cellular
products is essential
-Any disruption can cause new phenotypes
-Changes can be large scale or small scale
-Large scale: chromosomal changes
-Small scale: nucleotide substitutions, insertions, or deletions

point mutation

change a single nucleotide pair
of a gene

Substitution mutation

the replacement of one nucleotide and its partner with another pair of
nucleotides

silent mutation

change still codes for the same amino acid (remember: redundancy in the genetic
code)

missense mutation

change results in a different amino acid