AP Biology 6.3-6.4: Transcription and Translation

Central dogma of molecular biology

The central dogma describes the usual flow of genetic information: DNA is transcribed into RNA, and RNA is translated into protein. DNA stores the information, mRNA carries a copy, and ribosomes use that copy to assemble a polypeptide.

Why are proteins able to perform many different structures and functions?

Proteins are built from 20 common amino acids whose side chains differ in size, polarity, charge, and chemical reactivity. The amino acid sequence determines interactions and folding, producing diverse protein structures and functions.

Transcription

Transcription is the synthesis of an RNA strand using one DNA strand as a template. RNA polymerase catalyzes complementary base pairing and forms the RNA molecule in the $5' \to 3'$ direction.

Translation

Translation is the ribosome-mediated conversion of mRNA nucleotide information into a polypeptide amino acid sequence. The ribosome reads successive three-base codons and links the specified amino acids.

How does the nucleotide sequence of a gene determine a protein's amino acid sequence?

Transcription produces an mRNA sequence complementary to the DNA template. During translation, the ribosome reads the mRNA in three-nucleotide codons, and each codon specifies an amino acid or a termination signal.

Colinearity of gene expression

Genetic information is translated colinearly: the first three nucleotides of the coding mRNA specify the first amino acid, the next three specify the second, and so on. The order of nucleotides is preserved as the order of amino acids.

Codon

A codon is a sequence of three nucleotides in mRNA that specifies an amino acid or a stop signal during translation. Codons are read sequentially in a fixed reading frame.

Why must the genetic code use three-nucleotide codons rather than two-nucleotide codons?

With four possible RNA nucleotides, two-base combinations provide only $4^2 = 16$ possibilities, fewer than the 20 amino acids. Three-base combinations provide $4^3 = 64$ possibilities, enough to encode all amino acids plus stop signals.

Degeneracy of the genetic code

The genetic code is degenerate because most amino acids are specified by more than one codon. Codons for the same amino acid often differ at only one position, commonly the third nucleotide.

Start codon

AUG is the usual start codon. It establishes the translation reading frame and generally specifies methionine as the first amino acid in the newly synthesized polypeptide.

Stop codons

UAA, UAG, and UGA are stop codons. They do not specify amino acids; instead, they signal termination of translation and release of the polypeptide.

Why is the translation reading frame important?

The ribosome groups mRNA nucleotides into consecutive sets of three beginning at the start codon. Changing the starting position changes every downstream codon and therefore can drastically alter the resulting protein.

Frameshift mutation

A frameshift mutation results when nucleotide insertions or deletions change the mRNA sequence by a number not divisible by three. This shifts the reading frame, altering all subsequent codons and often producing a nonfunctional or prematurely terminated protein.

What is the typical effect of inserting or deleting three nucleotides in a coding sequence?

The reading frame is usually preserved because three nucleotides correspond to one codon. One amino acid may be added or removed while the downstream amino acid sequence remains aligned, although the protein can still be affected.

How can degeneracy reduce the effect of some mutations?

A nucleotide substitution may change a codon to another codon for the same amino acid, producing a silent mutation. Even when the amino acid changes, similar codons often specify chemically similar amino acids, which may lessen the effect on protein function.

Why does a one-nucleotide substitution sometimes have no effect on a protein?

Because the code is degenerate, the altered codon may still specify the same amino acid. Such a synonymous or silent substitution leaves the primary amino acid sequence unchanged, although effects on gene expression or RNA processing are possible in some contexts.

Why might a one-nucleotide substitution be less damaging when it changes an amino acid?

Codons that are similar often encode amino acids with similar chemical properties, such as similar charge or polarity. The resulting conservative substitution may preserve protein folding or function better than a chemically dissimilar substitution.

Universality of the genetic code

The genetic code is nearly universal, meaning that organisms generally use the same codons for the same amino acids. This conservation supports the idea that organisms share a common evolutionary origin and allows a gene from one organism to be expressed in another.

A bacterial mRNA has the sequence $5'-AUGGCUUAA-3'$. What peptide sequence is encoded?

Reading from the AUG start codon gives AUG-GCU-UAA. The sequence encodes methionine followed by alanine, then terminates at UAA.

Messenger RNA and noncoding RNA

Messenger RNA carries protein-coding information from DNA to ribosomes. Other DNA regions are transcribed into noncoding RNAs that function as RNA molecules rather than serving as templates for protein synthesis.

Template strand versus coding strand

The template strand is the DNA strand read by RNA polymerase and is complementary to the RNA transcript. The coding, or nontemplate, strand has nearly the same sequence as the RNA, except DNA contains T where RNA contains U.

How are DNA and RNA base-pairing rules related during transcription?

RNA polymerase uses the DNA template to add complementary RNA nucleotides: DNA A pairs with RNA U, DNA T with RNA A, DNA C with RNA G, and DNA G with RNA C. The RNA strand is synthesized antiparallel to the template.

In which directions are the DNA template read and the RNA transcript synthesized?

RNA polymerase reads the DNA template in the $3' \to 5'$ direction and synthesizes RNA in the $5' \to 3'$ direction. Nucleotides are added to the $3'$ end of the growing RNA strand.

How does transcription differ from DNA replication with respect to nucleotide composition?

Transcription produces RNA, so uracil is used in place of thymine. Unlike DNA replication, transcription generally copies only one DNA strand and produces a single-stranded RNA transcript rather than a complete DNA copy.

Transcription bubble

A transcription bubble is the locally unwound region of DNA where RNA polymerase separates the DNA strands and synthesizes RNA. DNA ahead of the enzyme unwinds, while DNA behind it rewinds.

What do upstream, downstream, and the +1 site mean in transcription?

The +1 site is the DNA position corresponding to the first nucleotide transcribed into RNA. Positions before it are upstream and receive negative numbering, whereas positions after it are downstream and receive positive numbering.

Promoter

A promoter is a DNA sequence, usually upstream of a gene, where transcription machinery binds and transcription begins. Its sequence helps determine whether and how frequently the gene is transcribed.

Regulatory sequences in a transcription unit

A transcription unit can include regulatory DNA sequences as well as the protein-coding sequence. Upstream regulatory regions help control transcription, while untranslated regions can occur before the coding sequence at the $5'$ end or after it at the $3'$ end.

A DNA coding strand contains $5'-ATGCCCTAA-3'$. What mRNA sequence corresponds to it?

The mRNA has the same sequence as the coding strand with U replacing T: $5'-AUGCCCUAA-3'$. It would encode methionine, proline, and then a stop signal.

A DNA template strand contains $3'-TACGGGATT-5'$. What mRNA sequence will RNA polymerase synthesize?

The complementary RNA is synthesized $5' \to 3'$ as $5'-AUGCCCUAA-3'$. The template is read $3' \to 5'$ while the RNA grows in the opposite direction.

What are the conserved promoter regions commonly recognized in many bacteria?

Many bacterial promoters contain consensus sequences near the $-10$ region, often TATAAT, and the $-35$ region, often TTGACA. These sequences are recognized by the sigma factor of RNA polymerase.

Why does the bacterial $-10$ promoter region help initiate transcription?

The $-10$ region is relatively rich in A and T, whose base pairs are held together by fewer hydrogen bonds than G-C pairs. This makes local DNA unwinding easier during transcription initiation.

Bacterial RNA polymerase holoenzyme

The bacterial holoenzyme consists of the RNA polymerase core enzyme plus a sigma factor. The core catalyzes RNA synthesis, while sigma recognizes promoter sequences and directs the polymerase to an appropriate initiation site.

What are the principal roles of the bacterial RNA polymerase subunits?

The two alpha subunits help assemble the enzyme on DNA, the beta subunit binds incoming ribonucleoside triphosphates, and the beta-prime subunit binds the DNA template. The sigma subunit provides promoter specificity during initiation.

What happens during initiation of prokaryotic transcription?

Sigma factor recognizes and binds the promoter, after which the core RNA polymerase joins and locally unwinds the DNA. The enzyme begins making short, abortive RNA transcripts before escaping the promoter and entering elongation.

Why is the sigma factor released after transcription initiation?

Sigma is primarily needed to recognize the promoter and establish the correct starting site. Once the RNA polymerase has begun elongating the transcript, the core enzyme can move along the template without sigma.

Prokaryotic transcription elongation

During elongation, the RNA polymerase core travels along the DNA template while adding ribonucleotides to the $3'$ end of the RNA.

Why can transcription and translation occur simultaneously in prokaryotes?

Prokaryotes lack a membrane-bound nucleus, so ribosomes can begin translating an mRNA while it is still being transcribed. Multiple polymerases and ribosomes can act concurrently, allowing rapid production of a protein.

Polycistronic mRNA

A polycistronic mRNA contains coding regions for more than one protein, a common arrangement in compact prokaryotic genomes. One transcript can therefore be translated into multiple different polypeptides.

Rho-dependent termination

In rho-dependent termination, the rho protein moves along the nascent RNA and catches a stalled RNA polymerase near the end of the gene. Its interaction with the polymerase releases the RNA transcript and ends transcription.

Rho-independent termination

Rho-independent termination involves RNA sequences that form a stable hairpin, followed by a weak RNA-DNA interaction in an A-U-rich region. The hairpin stalls RNA polymerase, and the weak interaction allows the transcript and polymerase to dissociate.

What is the role of general transcription factors in eukaryotic transcription initiation?

General transcription factors bind the promoter and help recruit and position RNA polymerase II. Together they form a transcription initiation complex, unwind the DNA near the start site, and enable RNA synthesis.

How does eukaryotic transcription initiation differ from bacterial initiation?

Eukaryotic RNA polymerase generally requires general transcription factors to recognize and assemble at a promoter, whereas bacterial sigma factor directs the RNA polymerase holoenzyme to the promoter. Eukaryotic transcription also occurs in the nucleus.

What happens during eukaryotic transcription elongation and termination?

During elongation, RNA polymerase II moves along the template strand and adds RNA nucleotides to the $3'$ end of the transcript. After transcribing a termination-related sequence, the RNA is cleaved and polymerase eventually dissociates from the DNA.

What is the purpose of the $5'$ cap on a eukaryotic pre-mRNA?

A modified guanine nucleotide is added to the $5'$ end of the pre-mRNA. The $5'$ cap helps protect the transcript from degradation, assists in nuclear export, and helps recruit the ribosome for translation.

What is the purpose of the poly-A tail on a eukaryotic pre-mRNA?

A stretch of adenine nucleotides is added to the $3'$ end of the RNA after cleavage. The poly-A tail increases mRNA stability, helps with nuclear export, and promotes efficient translation.

Introns and exons

Introns are transcribed regions that are removed from a eukaryotic pre-mRNA. Exons remain in the mature mRNA and are joined together before the transcript is translated.

RNA splicing

RNA splicing removes introns from a eukaryotic pre-mRNA and ligates the exons together. The spliceosome, composed of small nuclear RNAs and proteins, carries out this processing.

Why can alternative splicing increase protein diversity?

Alternative splicing allows different combinations of exons from the same pre-mRNA to be retained in mature mRNAs. These distinct mRNAs can be translated into different protein isoforms from one gene.

How is a mature eukaryotic mRNA prepared for translation?

The pre-mRNA is processed by addition of a $5'$ cap, removal of introns and joining of exons by splicing, and addition of a $3'$ poly-A tail. The mature mRNA is then exported from the nucleus to the cytoplasm.

What is the role of transfer RNA during translation?

Each tRNA carries a specific amino acid and contains an anticodon that base-pairs with a complementary mRNA codon. This adaptor function converts the nucleotide sequence of mRNA into an amino acid sequence.

How is a tRNA matched with its correct amino acid?

Aminoacyl-tRNA synthetases recognize particular tRNAs and use ATP to attach the corresponding amino acid. Accurate charging is essential because the ribosome checks codon-anticodon pairing but does not independently verify the attached amino acid.

Ribosome structure and function

A ribosome consists of small and large subunits made of ribosomal RNA and proteins. The small subunit helps position and read the mRNA, while the large subunit catalyzes peptide-bond formation.

What are the A, P, and E sites of a ribosome?

The A site binds the incoming aminoacyl-tRNA, the P site holds the tRNA attached to the growing polypeptide, and the E site is where the uncharged tRNA exits. During elongation, tRNAs move through the sites in the order A, P, then E.

What happens during translation initiation?

The small ribosomal subunit binds the mRNA and identifies the start codon. The initiator tRNA carrying methionine pairs with AUG in the P site, and the large subunit joins to form the complete initiation complex.

What are the three repeated steps of translation elongation?

An aminoacyl-tRNA enters the A site and pairs with the codon, the ribosome forms a peptide bond and transfers the growing chain to that tRNA, and translocation moves the ribosome one codon along the mRNA. The uncharged tRNA then exits through the E site.

In which direction are mRNA and polypeptides read or synthesized during translation?

The ribosome moves along mRNA from $5' \to 3'$. The polypeptide is synthesized from its amino-terminal ($N$-terminal) end toward its carboxyl-terminal ($C$-terminal) end.

How does translation terminate?

When a stop codon enters the A site, no tRNA pairs with it. A release factor binds instead, catalyzes release of the completed polypeptide from the tRNA, and causes the ribosomal subunits and mRNA to dissociate.

Why can multiple ribosomes translate one mRNA simultaneously?

After one ribosome moves away from the start codon, another can bind and begin translation. A group of ribosomes translating the same mRNA is called a polyribosome or polysome, allowing many copies of a protein to be produced rapidly.