Transcription and Translation

The Relationship
Between Genes and
Proteins

Table of Contents
History: linking genes and proteins
Getting from gene to protein: transcription

Evidence for mRNA

Overview of transcription
RNA polymerase
Stages of Transcription
Promoter recognition
Chain initiation
Chain elongation
Chain termination

mRNA Synthesis/Processing
References

Table of Contents (continued)

Getting from gene to protein: genetic code
Getting from gene to protein: translation

Translation Initiation
Translation Elongation
Translation Termination
References

History: linking genes and proteins

1900s Archibald Garrod

Inborn errors of metabolism: inherited human metabolic diseases

(more information)
Genes are the inherited factors
Enzymes are the biological molecules that drive
metabolic reactions
Enzymes are proteins
Question:
How do the inherited factors, the genes, control the structure and
activity of enzymes (proteins)?

History: linking genes and proteins

Beadle and Tatum (1941) PNAS USA 27, 499506.
Hypothesis:

If genes control structure and activity of metabolic enzymes, then

mutations in genes should disrupt production of required nutrients,
and that disruption should be heritable.

Method:

Isolated ~2,000 strains from single irradiate spores (Neurospora)

that grew on rich but not minimal medium. Examples: defects in B1,
B6 synthesis.

Conclusion:

Genes govern the ability to synthesize amino acids, purines and

vitamins.

History: linking genes and proteins

1950s: sickle-cell anemia

Glu to Val change in hemoglobin

Sequence of nucleotides in gene determines sequence of amino
acids in protein
Single amino acid change can alter the function of the protein

Tryptophan synthase gene in E. coli

Mutations resulted in single amino acid change

Order of mutations in gene same as order of affected amino acids

From gene to protein: transcription

Gene sequence (DNA) recopied or transcribed to RNA
sequence
Product of transcription is a messenger molecule that
delivers the genetic instructions to the protein synthesis
machinery: messenger RNA (mRNA)

Transcription: evidence for mRNA

Brenner, S., Jacob, F. and Meselson, M. (1961) Nature
190, 57681.
Question: How do genes work?

Does each one encode a different type of ribosome which in turn

synthesizes a different protein, OR
Are all ribosomes alike, receiving the genetic information to create
each different protein via some kind of messenger molecule?

Transcription: evidence for mRNA

E. coli cells switch from making bacterial proteins to
phage proteins when infected with bacteriophage T4.
Grow bacteria on medium containing heavy nitrogen
(15N) and carbon (13C).
Infect with phage T4.
Immediately transfer to light medium containing
radioactive uracil.

Transcription: evidence for mRNA

If genes encode different ribosomes, the newly
synthesized phage ribosomes will be light.
If genes direct new RNA synthesis, the RNA will contain
radiolabeled uracil.
Results:

Ribosomes from phage-infected cells were heavy, banding at the

same density on a CsCl gradient as the original ribosomes.
Newly synthesized RNA was associated with the heavy ribosomes.
New RNA hybridized with viral ssDNA, not bacterial ssDNA.

Transcription: evidence for mRNA

Conclusion

Expression of phage DNA results in new phage-specific RNA

molecules (mRNA)
These mRNA molecules are temporarily associated with ribosomes
Ribosomes do not themselves contain the genetic directions for
assembling individual proteins

Transcription: overview
Transcription requires:
ribonucleoside 5 triphosphates:

ATP, GTP, CTP and UTP

bases are adenine, guanine, cytosine and uracil
sugar is ribose (not deoxyribose)

DNA-dependent RNA polymerase

Template (sense) DNA strand
Animation of transcription

Transcription: overview
Features of transcription:
RNA polymerase catalyzes sugar-phosphate bond
between 3-OH of ribose and the 5-PO4.
Order of bases in DNA template strand determines order
of bases in transcript.
Nucleotides are added to the 3-OH of the growing chain.
RNA synthesis does not require a primer.

Transcription: overview
In prokaryotes transcription and translation are coupled.
Proteins are synthesized directly from the primary
transcript as it is made.
In eukaryotes transcription and translation are separated.
Transcription occurs in the nucleus, and translation occurs
in the cytoplasm on ribosomes.
Figure comparing eukaryotic and prokaryotic transcription
and translation.

Transcription: RNA Polymerase

DNA-dependent

DNA template, ribonucleoside 5 triphosphates, and Mg2+

Synthesizes RNA in 5 to 3 direction

E. coli RNA polymerase consists of 5 subunits
Eukaryotes have three RNA polymerases

RNA polymerase II is responsible for transcription of protein-coding

genes and some snRNA molecules
RNA polymerase II has 12 subunits
Requires accessory proteins (transcription factors)
Does not require a primer

Stages of Transcription
Promoter Recognition
Chain Initiation
Chain Elongation
Chain Termination

Transcription: promoter recognition

Transcription factors bind to promoter sequences and
recruit RNA polymerase.
DNA is bound first in a closed complex. Then, RNA
polymerase denatures a 1215 bp segment of the DNA
(open complex).
The site where the first base is incorporated into the
transcription is numbered +1 and is called the
transcription start site.
Transcription factors that are required at every promoter
site for RNA polymerase interaction are called basal
transcription factors.

Promoter recognition: promoter sequences

Promoter sequences vary considerably.
RNA polymerase binds to different promoters with
different strengths; binding strength relates to the level of
gene expression
There are some common consensus sequences for
promoters:

Example: E. coli 35 sequence (found 35 bases 5 to the start of

transcription)
Example: E. coli TATA box (found 10 bases 5 to the start of
transcription)

Promoter recognition: enhancers

Eukaryotic genes may also have enhancers.
Enhancers can be located at great distances from the
gene they regulate, either 5 or 3 of the transcription
start, in introns or even on the noncoding strand.
One of the most common ways to identify promoters and
enhancers is to use a reporter gene.

Promoter recognition: other players

Many proteins can regulate gene expression by
modulating the strength of interaction between the
promoter and RNA polymerase.
Some proteins can activate transcription (upregulate gene
expression).
Some proteins can inhibit transcription by blocking
polymerase activity.
Some proteins can act both as repressors and activators
of transcription.

Transcription: chain initiation

Chain initiation:
RNA polymerase locally denatures the DNA.
The first base of the new RNA strand is placed
complementary to the +1 site.
RNA polymerase does not require a primer.
The first 8 or 9 bases of the transcript are linked.
Transcription factors are released, and the polymerase
leaves the promoter region.
Figure of bacterial transcription initiation.

Transcription: chain elongation

Chain elongation:
RNA polymerase moves along the transcribed or template
DNA strand.
The new RNA molecule (primary transcript) forms a short
RNA-DNA hybrid molecule with the DNA template.

Transcription: chain termination

Most known about bacterial chain termination
Termination is signaled by a sequence that can form a
hairpin loop.
The polymerase and the new RNA molecule are released
upon formation of the loop.
Review the transcription animation.

Transcription: mRNA synthesis/processing

Prokaryotes: mRNA transcribed directly from DNA
template and used immediately in protein synthesis
Eukaryotes: primary transcript must be processed to
produce the mRNA

Noncoding sequences (introns) are removed

Coding sequences (exons) spliced together
5-methylguanosine cap added
3-polyadenosine tail added

Transcription: mRNA synthesis/processing

Removal of introns and splicing of exons can occur
several ways

For introns within a nuclear transcript, a spliceosome is required.

Splicesomes protein and small nuclear RNA (snRNA)
Specificity of splicing comes from the snRNA, some of which contain
sequences complementary to the splice junctions between introns and
exons

Alternative splicing can produce different forms of a protein from

the same gene
Mutations at the splice sites can cause disease
Thalassemia

Breast cancer (BRCA 1)

Transcription: mRNA synthesis/processing

RNA splicing inside the nucleus on particles called
spliceosomes.
Splicesomes are composed of proteins and small RNA
molecules (100200 bp; snRNA).
Both proteins and RNA are required, but some suggesting
that RNA can catalyze the splicing reaction.
Self-splicing in Tetrahymena: the RNA catalyzes its own
splicing
Catalytic RNA: ribozymes

From gene to protein: genetic code

Central Dogma

Information travels from DNA to RNA to Protein

Is there a one-to-one correspondence between DNA, RNA and Protein?
DNA and RNA each have four nucleotides that can form them; so yes, there
is a one-to-one correspondence between DNA and RNA.
Proteins can be composed of a potential 20 amino acids; only four RNA
nucleotides: no one-to-one correspondence.
How then does RNA direct the order and number of amino acids in a protein?

From gene to protein: genetic code

How many bases are required for each amino acid?
(4 bases)2bases/aa = 16 amino acidsnot enough
(4 bases)3bases/aa = 64 amino acid possibilities

Minimum of 3 bases/aa required

What is the nature of the code?

Does it have punctuation? Is it overlapping?

Crick, F.H. et al. (1961) Nature 192, 122732.
(http://profiles.nlm.nih.gov/SC/B/C/B/J/ )
3-base, nonoverlapping code that is read from a fixed point.

From gene to protein: genetic code

Nirenberg and Matthaei: in vitro protein translation

Found that adding rRNA prolonged cell-free protein synthesis

Adding artificial RNA synthesized by polynucleotide phosphorylase
(no template, UUUUUUUUU) stimulated protein synthesis more
The protein that came out of this reaction was polyphenylalanine
(UUU = Phe)
Other artificial RNAs: AAA = Lys; CCC =Pro

From gene to protein: genetic code

Nirenberg:

Triplet binding assay: add triplet RNA, ribosomes, binding factors,

GTP, and radiolabeled charged tRNA (figure)
UUU trinucleotide binds to Phe-tRNA
UGU trinucleotide binds to CYS-tRNA

By fits and starts the triplet genetic code was worked out.
Each three-letter word (codon) specifies an amino acid or
directions to stop translation.
The code is redundant or degenerate: more than one way to
encode an amino acid

From gene to protein: Translation

Components required for translation:

mRNA
Ribosomes
tRNA
Aminoacyl tRNA synthetases
Initiation, elongation and termination factors

Animation of translation

Translation: initiation
Ribosome small subunit binds to mRNA
Charged tRNA anticodon forms base pairs with the mRNA
codon
Small subunit interacts with initiation factors and special
initiator tRNA that is charged with methionine
mRNA-small subunit-tRNA complex recruits the large
subunit
Eukaryotic and prokaryotic initiation differ slightly

Translation: initiation
The large subunit of the ribosome contains three binding
sites

Amino acyl (A site)

Peptidyl (P site)
Exit (E site)

At initiation,

The tRNAfMet occupies the P site

A second, charged tRNA complementary to the next codon binds
the A site.

Translation: elongation
Elongation
Ribosome translocates by three bases after peptide bond
formed
New charged tRNA aligns in the A site
Peptide bond between amino acids in A and P sites is
formed
Ribosome translocates by three more bases
The uncharged tRNA in the A site is moved to the E site.

Translation: elongation
EF-Tu

recruits charged tRNA to A site. Requires

hydrolysis of GTP
Peptidyl transferase catalyzes peptide bond formation
(bond between aa and tRNA in the P site converted to
peptide bond between the two amino acids)
Peptide bond formation requires RNA and may be a
ribozyme-catalyzed reaction

Translation: termination
Termination
Elongation proceeds until STOP codon reached

UAA, UAG, UGA

No tRNA normally exists that can form base pairing with a

STOP codon; recognized by a release factor
tRNA charged with last amino acid will remain at P site
Release factors cleave the amino acid from the tRNA
Ribosome subunits dissociate from each other
Review the animation of translation