BIS 1A Handout 17 - From Gene to Protein

The Flow of Genetic Information

The information content of DNA is in the form of specific sequences of nucleotides along the DNA strands.

Gene expression, the process by which DNA directs protein synthesis, includes two stages called transcription and translation.

Proteins are the links between genotype and phenotype.

Genes specify proteins via transcription and translation

Transcription and translation are the two main processes linking gene to protein.

Genes provide the instructions for making specific proteins.

The bridge between DNA and protein synthesis is the nucleic acid RNA.

RNA is chemically similar to DNA, except that it contains ribose as its sugar and substitutes the nitrogenous base uracil for thymine.

An RNA molecule almost always consists of a single strand.

To get from DNA to protein requires two major stages: transcription and translation.

During transcription, a DNA strand provides a template for the synthesis of a complementary RNA strand.

Just as a DNA strand provides a template for the synthesis of each new complementary strand during DNA replication, it provides a template for assembling a sequence of RNA nucleotides.

Transcription of many genes produces a messenger RNA (mRNA) molecule.

During translation, the genetic code is translated to a sequence of amino acids.

The site of translation is the ribosome, complex particles that facilitate the orderly assembly of amino acids into polypeptide chains.

Why can't proteins be translated directly from DNA?

The use of an RNA intermediate provides protection for DNA and its genetic information.

Using an RNA intermediate allows more copies of a protein to be made simultaneously, since many RNA transcripts can be made from one gene.

Also, each gene transcript can be translated repeatedly.

The basic mechanics of transcription and translation are similar in eukaryotes and prokaryotes.

Because bacteria lack nuclei, their DNA is not segregated from ribosomes and other protein-synthesizing equipment.

In a eukaryotic cell, transcription occurs in the nucleus, and translation occurs at ribosomes in the cytoplasm.

The transcription of a protein-coding eukaryotic gene results in pre-mRNA.

The initial RNA transcript of any gene is called a primary transcript.

RNA processing yields the finished mRNA.

In the genetic code, nucleotide triplets specify amino acids.

Triplets of nucleotide bases are the smallest units of uniform length that can code for all the amino acids.

With a triplet code, three consecutive bases specify an amino acid, creating 43 (64) possibilities.

During transcription, one DNA strand, the template strand, provides a template for ordering the sequence of nucleotides in an RNA transcript.

A given DNA strand can be the template strand for some genes along a DNA molecule, while for other genes in other regions, the complementary strand may function as the template.

The complementary RNA molecule is synthesized according to base-pairing rules, except that uracil is the complementary base to adenine.

Like a new strand of DNA, the RNA molecule is synthesized in an antiparallel direction to the template strand of DNA.

The mRNA base triplets are called codons, and they are written in the 5' ( 3' direction.

During translation, the sequence of codons along an mRNA molecule is translated into a sequence of amino acids making up the polypeptide chain.

During translation, the codons are read in the 5' ( 3' direction along the mRNA.

Sixty-one of 64 triplets code for amino acids.

Several codons may specify the same amino acid, but no codon specifies more than one amino acid.

The codon AUG not only codes for the amino acid methionine, but also indicates the "start" of translation.

Three codons do not indicate amino acids but are "stop" signals marking the termination of translation.

The genetic code must have evolved very early in the history of life.

The genetic code is nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals.

In laboratory experiments, genes can be transcribed and translated after they are transplanted from one species to another.

This has permitted bacteria to be programmed to synthesize certain human proteins after insertion of the appropriate human genes.

A shared genetic vocabulary is a reminder of the kinship that bonds all life on Earth.

Transcription is the DNA-directed synthesis of RNA: a closer look

Messenger RNA, the carrier of information from DNA to the cell's protein-synthesizing machinery, is transcribed from the template strand of a gene.

RNA polymerase separates the DNA strands at the appropriate point and bonds the RNA nucleotides as they base-pair along the DNA template.

Like DNA polymerases, RNA polymerases can only assemble a polynucleotide in its 5' ( 3' direction.

Unlike DNA polymerases, RNA polymerases don't need a primer.

Specific sequences of nucleotides along the DNA mark where gene transcription begins and ends.

RNA polymerase attaches and initiates transcription at the promoter.

In prokaryotes, the sequence that signals the end of transcription is called the terminator.

Molecular biologists refer to the direction of transcription as "downstream" and the other direction as "upstream."

Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules.

In contrast, eukaryotes have three RNA polymerases (I, II, and III) in their nuclei.

RNA polymerase II is used for mRNA synthesis.

Transcription can be separated into three stages: initiation, elongation, and termination of the RNA chain.

The presence of a promoter sequence determines which strand of the DNA helix is the template.

A crucial promoter DNA sequence is called a TATA box.

Within the promoter is the starting point for the transcription of a gene.

The promoter also includes a binding site for RNA polymerase several dozen nucleotides "upstream" of the start point.

In prokaryotes, RNA polymerase can recognize and bind directly to the promoter region.

In eukaryotes, proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription.

Only after certain transcription factors are attached to the promoter does RNA polymerase II bind to it.

The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex.

RNA polymerase then starts transcription.

Transcription progresses at a rate of 60 nucleotides per second in eukaryotes.

A single gene can be transcribed simultaneously by several RNA polymerases at a time.

Transcription proceeds until after the RNA polymerase transcribes a terminator sequence in the DNA.

In prokaryotes, RNA polymerase stops transcription right at the end of the terminator.

Both the RNA and DNA are then released.

In eukaryotes, the pre-mRNA is cleaved from the growing RNA chain while RNA polymerase II continues to transcribe the DNA.

First, the polymerase transcribes a DNA sequence called the polyadenylation signal.

At a point about 10 to 35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme.

The polymerase continues transcribing for hundreds of nucleotides.

Transcription is terminated when the polymerase eventually falls off the DNA.

Eukaryotic cells modify RNA after transcription

Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm.

At the 5' end of the pre-mRNA molecule, a modified form of guanine is added, the 5' cap.

At the 3' end, an enzyme adds 50 to 250 adenine nucleotides, the poly-A tail.

These modifications share several important functions.

They seem to facilitate the export of mRNA from the nucleus.

They help protect mRNA from hydrolytic enzymes.

They help the ribosomes attach to the 5' end of the mRNA.

The most remarkable stage of RNA processing occurs during the removal of a large portion of the RNA molecule in a cut-and-paste job of RNA splicing.

Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides.

Noncoding segments of nucleotides called intervening regions, or introns, lie between coding regions.

The final mRNA transcript includes coding regions, exons, which are translated into amino acid sequences, plus the leader and trailer sequences.

RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence.

This splicing is accomplished by a spliceosome.

Spliceosomes are located in the nucleus and consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites.

Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA).

The spliceosome interacts with certain sites along an intron, releasing the introns and joining together the two exons that flanked the introns.

snRNAs appear to play a major role in catalytic processes, as well as spliceosome assembly and splice site recognition.

Introns and RNA splicing appear to have several functions.

Some introns play a regulatory role in the cell. These introns contain sequences that control gene activity in some way.

Splicing itself may regulate the passage of mRNA from the nucleus to the cytoplasm.

One clear benefit of split genes is to enable one gene to encode for more than one polypeptide.

Alternative RNA splicing gives rise to two or more different polypeptides, depending on which segments are treated as exons.

Sex differences in fruit flies may be due to differences in splicing RNA transcribed from certain genes.

Proteins often have a modular architecture with discrete structural and functional regions called domains.

The presence of introns in a gene may facilitate the evolution of new and potentially useful proteins as a result of a process known as exon shuffling.

In many cases, different exons code for different domains of a protein.

Translation is the RNA-directed synthesis of a polypeptide: a closer look

In the process of translation, a cell interprets a series of codons along an mRNA molecule and builds a polypeptide.

The interpreter is transfer RNA (tRNA), which transfers amino acids to a ribosome.

The ribosome adds each amino acid carried by tRNA to the growing end of the polypeptide chain.

Each tRNA arriving at the ribosome carries a specific amino acid at one end and has a specific nucleotide triplet, an anticodon, at the other.

The anticodon base-pairs with a complementary codon on mRNA.

If the codon on mRNA is UUU, a tRNA with an AAA anticodon and carrying phenylalanine will bind to it.

Like other types of RNA, tRNA molecules are transcribed from DNA templates in the nucleus.

Once it reaches the cytoplasm, each tRNA picks up its designated amino acid, deposits the amino acid at the ribosome, and recycles to pick up another copy of that amino acid.

If each anticodon had to be a perfect match to each codon, we would expect to find 61 types of tRNA, but the actual number is about 45.

The anticodons of some tRNAs recognize more than one codon.

This is possible because the rules for base pairing between the third base of the codon and anticodon are relaxed (called wobble).

At the wobble position, U on the anticodon can bind with A or G in the third position of a codon.

Wobble explains why the synonymous codons for a given amino acid can differ in their third base, but not usually in their other bases.

Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase.

There is a different synthetase to match each of the 20 different amino acids.

Ribosomes facilitate the specific coupling of the tRNA anticodons with mRNA codons during protein synthesis.

Each ribosome is made up of a large and a small subunit.

The subunits are composed of proteins and ribosomal RNA (rRNA).

In eukaryotes, the subunits are made in the nucleolus.

The subunits exit the nucleus via nuclear pores.

The large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule.

While very similar in structure and function, prokaryotic and eukaryotic ribosomes have enough differences that certain antibiotic drugs (like tetracycline) can inhibit prokaryotic ribosomes without inhibiting eukaryotic ribosomes.

Each ribosome has a binding site for mRNA and three binding sites for tRNA molecules.

The A site carries the tRNA with the next amino acid to be added to the chain.

The P site holds the tRNA carrying the growing polypeptide chain.

Discharged tRNAs leave the ribosome at the E (exit) site.

The ribosome holds the tRNA and mRNA in close proximity and positions the new amino acid for addition to the carboxyl end of the growing polypeptide.

It then catalyzes the formation of the peptide bond.

The rRNA is the catalyst for peptide bond formation, not the protein.

Translation can be divided into three stages: initiation, elongation, and termination.

All three phases require protein "factors" that aid in the translation process.

Both initiation and chain elongation require energy provided by the hydrolysis of GTP.

Initiation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits.

First, a small ribosomal subunit binds with mRNA and a special initiator tRNA, which carries methionine and attaches to the start codon.

The small subunit then moves downstream along the mRNA until it reaches the start codon, AUG, which signals the start of translation.

This establishes the reading frame for the mRNA.

The initiator tRNA, already associated with the complex, then hydrogen-bonds with the start codon.

Proteins called initiation factors bring in the large subunit so that the initiator tRNA occupies the P site.

Elongation involves the participation of several protein elongation factors, and consists of a series of three-step cycles as each amino acid is added to the proceeding one.

During codon recognition, an elongation factor assists hydrogen bonding between the mRNA codon under the A site with the corresponding anticodon of tRNA carrying the appropriate amino acid.

This step requires the hydrolysis of two GTP.

During peptide bond formation, an rRNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site.

This step separates the tRNA at the P site from the growing polypeptide chain and transfers the chain, now one amino acid longer, to the tRNA at the A site.

During translocation, the ribosome moves the tRNA with the attached polypeptide from the A site to the P site.

Because the anticodon remains bonded to the mRNA codon, the mRNA moves along with it.

The next codon is now available at the A site.

The tRNA that had been in the P site is moved to the E site and then leaves the ribosome.

Translocation is fueled by the hydrolysis of GTP.

Effectively, translocation ensures that the mRNA is "read" 5' ( 3' codon by codon.

• The three steps of elongation continue to add amino acids codon by codon until the polypeptide chain is completed.

Termination occurs when one of the three stop codons reaches the A site.

A release factor binds to the stop codon and hydrolyzes the bond between the polypeptide and its tRNA in the P site.

This frees the polypeptide, and the translation complex disassembles.

Typically a single mRNA is used to make many copies of a polypeptide simultaneously.

Multiple ribosomes, polyribosomes, may trail along the same mRNA.

Polyribosomes can be found in prokaryotic and eukaryotic cells.

A ribosome requires less than a minute to translate an average-sized mRNA into a polypeptide.

During and after synthesis, a polypeptide coils and folds to its three-dimensional shape spontaneously.

Chaperone proteins may aid correct folding.

In addition, proteins may require posttranslational modifications before doing their particular job.

This may require additions such as sugars, lipids, or phosphate groups to amino acids.

Enzymes may remove some amino acids or cleave whole polypeptide chains.

Two or more polypeptides may join to form a protein.

Signal peptides target some eukaryotic polypeptides to specific destinations in the cell.

Two populations of ribosomes, free and bound, are active participants in protein synthesis.

Free ribosomes are suspended in the cytosol and synthesize proteins that reside in the cytosol.

Bound ribosomes are attached to the cytosolic side of the endoplasmic reticulum.

They synthesize proteins of the endomembrane system as well as proteins secreted from the cell.

Translation in all ribosomes begins in the cytosol, but a polypeptide destined for the endomembrane system or for export has a specific signal peptide region near the leading end.

A signal recognition particle (SRP) binds to the signal peptide and attaches it and its ribosome to a receptor protein in the ER membrane.

The SRP consists of a protein-RNA complex.

After binding, the SRP leaves and protein synthesis resumes with the growing polypeptide snaking across the membrane into the cisternal space via a protein pore.

An enzyme usually cleaves the signal polypeptide.

Secretory proteins are released entirely into the cisternal space, but membrane proteins remain partially embedded in the ER membrane.

Other kinds of signal peptides are used to target polypeptides to mitochondria, chloroplasts, the nucleus, and other organelles that are not part of the endomembrane system.

In these cases, translation is completed in the cytosol before the polypeptide is imported into the organelle.

Comparing gene expression in prokaryotes and eukaryotes reveals key differences

Although prokaryotes and eukaryotes carry out transcription and translation in very similar ways, they do have differences in cellular machinery and in details of the processes.

Eukaryotic RNA polymerases differ from those of prokaryotes and require transcription factors.

They differ in how transcription is terminated.

Their ribosomes also are different.

One major difference is that prokaryotes can transcribe and translate the same gene simultaneously. They have no nucleus.

The new protein quickly diffuses to its operating site.

In eukaryotes, the nuclear envelope segregates transcription from translation.

In addition, extensive RNA processing is carried out between these processes.

This provides additional steps whose regulation helps coordinate the elaborate activities of a eukaryotic cell.

Point mutations can affect protein structure and function

Mutations are changes in the genetic material.

A chemical change in just one base pair of a gene causes a point mutation.

If these occur in gametes or cells producing gametes, they may be transmitted to future generations.

For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin.

A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein.

A point mutation that results in the replacement of a pair of complementary nucleotides with another nucleotide pair is called a base-pair substitution.

Some base-pair substitutions have little or no impact on protein function.

In silent mutations, altered nucleotides still code for the same amino acids because of redundancy in the genetic code.

Other changes lead to switches from one amino acid to another with similar properties.

Still other mutations may occur in a region where the exact amino acid sequence is not essential for function.

Other base-pair substitutions cause a readily detectable change in a protein.

These are usually detrimental but can occasionally lead to an improved protein or one with novel capabilities.

Changes in amino acids at crucial sites, especially active sites, are likely to impact function.

Missense mutations are those that still code for an amino acid but a different one.

Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein.

Insertions and deletions are additions or losses of nucleotide pairs in a gene.

These affect protein structure more often than substitutions do.

Unless insertion or deletion mutations occur in multiples of three, they cause a frameshift mutation.

All the nucleotides downstream of the frameshift will be improperly grouped into codons.

The result will be extensive missense.

Mutations can occur in a number of ways.

Errors during DNA replication, DNA repair, or DNA recombination are called spontaneous mutations.

Rough estimates suggest that about 1 nucleotide in every 1010 is altered and inherited by daughter cells.

Mutagens are chemical or physical agents that interact with DNA to cause mutations.

Physical agents include high-energy radiation like X-rays and ultraviolet light.

Chemical mutagens fall into several categories.

Some chemicals are base analogues that may be substituted into DNA, but they pair incorrectly during DNA replication.

Other mutagens interfere with DNA replication by inserting into DNA and distorting the double helix.

Still others cause chemical changes in bases that change their pairing properties.

What is a gene? We revisit the question.

The Mendelian concept of a gene views it as a discrete unit of inheritance that affects phenotype.

Morgan and his colleagues assigned genes to specific loci on chromosomes.

We can also view a gene as a specific nucleotide sequence along a region of a DNA molecule.

We can define a gene functionally as a DNA sequence that codes for a specific polypeptide chain.

Even the one gene-one polypeptide definition must be refined and applied selectively.

Most eukaryotic genes contain large introns that have no corresponding segments in polypeptides.

Promoters and other regulatory regions of DNA are not transcribed either, but they must be present for transcription to occur.

Our molecular definition must also include the various types of RNA that are not translated into polypeptides, such as rRNA, tRNA, and other RNAs.

This is our definition of a gene: A gene is a region of DNA whose final product is either a polypeptide or an RNA molecule.