Gene to Protein: Transcription and Translation

From Gene to Protein

• Gene expression describes the process of creating a gene product
• This requires two steps: transcription and translation.
• Transcription copies DNA into RNA.
• Translation reads the RNA copy to assemble a protein.

Gene Expression

• Transcription involves copying genetic messages in DNA into RNA
• It is catalyzed by RNA polymerase.
• RNA polymerase reads the template strand in the 3' to 5' direction
• It synthesizes RNA from 5' to 3'.

Molecular Components of Transcription

• RNA polymerase separates DNA strands.
• It bonds RNA nucleotides by base-pairing along the DNA template.
• RNA polymerases assemble a polynucleotide in the 5' to 3' direction, similar to DNA polymerases.
• RNA polymerases can start a chain from scratch and do not need a primer, unlike DNA polymerases.

Types of RNA Polymerases

• Eukaryotes possess three RNA polymerases (I, II, and III).
• RNA polymerase I, located in the nucleolus, synthesizes ribosomal RNA (rRNA) precursors.
• RNA polymerases II and III reside in the nucleoplasm.
• RNA polymerase II synthesizes heterogeneous nuclear RNA (hnRNA) and small nuclear RNA (snRNA).
• RNA polymerase III synthesizes precursors to transfer RNAs (tRNAs), 5S rRNA, and other small RNA molecules.
• Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules.

Gene Structure

• A protein-coding gene includes a promoter, coding sequence, and terminator.
• The promoter is a base-pair sequence that indicates where transcription should start.
• The coding sequence comprises base pairs with coding information for the polypeptide chain.
• The terminator is a sequence that specifies where the mRNA transcript should end.
• The terminator contains the stop codons UAA, UAG and UGA

Synthesis of an RNA Transcript

• Transcription happens in three stages: initiation, elongation, and termination.
• Initiation involves RNA polymerase binding to the promoter, local DNA melting, and the formation of the first few phosphodiester bonds.
• During elongation, RNA polymerase joins ribonucleotides in the 5' to 3' direction to create the rest of the RNA molecule.
• Termination includes RNA polymerase and the RNA product dissociating from the DNA template.

Initiation

• The RNA polymerase first recognizes and binds to a promoter region
• The presence of a promoter sequence determines which strand of the DNA helix becomes the template.
• Promoters include a binding site for RNA polymerase.
• The initial RNA molecule, known as the primary transcript, requires post-transcriptional modification before export to the cytoplasm for translation.
• mRNA is ready for translation following transcription and post-transcriptional processing.

Elongation

• During elongation, the polymerase moves downstream, lengthening the RNA transcript in the 5' to 3' direction.

Termination

• Eventually, the RNA transcript is released, and the polymerase detaches from the DNA.
• In eukaryotes, transcription factors mediate the binding of RNA polymerase and the initiation of transcription.
• A completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex.
• A crucial promoter DNA sequence used in this process is the TATA box.

Transcription Elongation Details

• RNA polymerase starts transcription.
• As it moves, RNA polymerase untwists the double helix 10 to 20 bases at a time.
• The enzyme adds nucleotides to the 3' end of the growing strand.
• Behind the point of RNA synthesis, the double helix re-forms, and the RNA molecule peels away.
• Eukaryotic transcription progresses at 60 nucleotides per second.
• A single gene can be simultaneously transcribed by multiple RNA polymerases.
• The length of each new strand shows how far along the template the enzyme has traveled.
• The congregation of many polymerase molecules transcribing a single gene increases the amount of mRNA.
• This process helps the cell make the encoded protein in large amounts.

Termination of Transcription

• Transcription continues until RNA polymerase transcribes a terminator sequence in the DNA, signaling the end.
• In prokaryotes, RNA polymerase stops transcription at the terminator's end.
• In eukaryotes, the pre-mRNA is cleaved from the growing RNA chain, while RNA polymerase II continues transcribing the DNA.
• Specifically, polymerase transcribes a DNA sequence, the polyadenylation signal sequence, which codes for a polyadenylation sequence (AAUAAA) in the pre-mRNA
• The pre-mRNA is cut about 10 to 35 nucleotides downstream from this sequence.
• The polymerase continues transcribing for hundreds of nucleotides before transcription terminates when the polymerase falls off the DNA.

Eukaryotic Cells Modify RNA after Transcription

• Enzymes in the eukaryotic nucleus alter pre-mRNA so genetic messages can be dispatched to the cytoplasm.
• During RNA processing, both ends of the primary transcript are usually altered.
• Certain interior parts of the molecule are cut out, and the remaining parts are spliced together.

RNA Processing

• A modified form of guanine is added to the 5' end
• 50 to 250 adenine nucleotides are added to the 3' end.

5' Cap

• A 5' methylguanosine cap forms quickly after RNA synthesis begins.
• The last of the three phosphates is removed by an enzyme, resulting in diphosphate.
• GMP (Guanosine monophosphate) is added in an inverted orientation so guanosine 5' end faces 5' end of RNA chain.
• Guanosine is methylated at position 7 on guanine base, while the nucleotide on the triphosphate bridge internal side is methylated at ribose 2' position (methylguanosine cap). The enzyme Guanylyltransferase enacts this.

Functions of the 5' cap

• May prevent exonuclease digestion of mRNA 5' end.
• Aids in mRNA transport out of the nucleus.
• Plays an important role in the initiation of mRNA translation.

Polyadenylation

• Most mRNAs contain a string of 50-250 adenosine residues at the 3' end, which forms a poly(A) tail.
• This tail protects the mRNA from degradation and helps it move from the nucleus to the cytoplasm.
• A signal ~20 nt upstream from the poly(A) addition site indicates where to add poly(A)
• Poly(A) polymerase, poly(A) binding proteins, and several cleavage factors are involved in this process.

mRNA Splicing

• Requires breaks at 5' and 3' intron ends
• It also covalently joins adjacent exons (ligation) via splice sites, which is carried out by spliceosomes
• Introns may have disadvantages, such as extra DNA and energy needed for processing and replication.
• Introns may have advantages due to their modular design allowing for greater variation and easier introduction of that variation, thereby producing more than one protein.

mRNA Splicing Details

• Sequences adjacent to introns contain preferred nucleotides that play an important role in splice site recognition.
• Splicing must be very precise.
• The most common conserved sequence at eukaryotic exon-intron borders in mammalian pre-mRNA is G/GU at the 5' intron end (5' splice site) and AG/G at the 3' end (3' splice site).

Modifications Functions

• These modifications facilitate the export of mRNA from the nucleus.
• They protect mRNA from hydrolytic enzymes.
• Help ribosomes attach to the 5' end of the mRNA.

Alteration of mRNA Ends

• This results in Ends modifications, where the 5' end receives a modified nucleotide cap, and the 3' end gets a poly-A tail

RNA Splicing

• This results in internal modifications.
• Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides.
• Noncoding segments of nucleotides (introns) lie between coding regions (exons).
• RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence.

The Final mRNA Transcript

• It includes coding regions, which translate into amino acid sequences, and two non-coding regions.
• The non-coding regions are at each end and are called UTRs (Untranslated regions).
• Splicing is carried out by spliceosomes.

Spliceosomes

• They consist of a variety of proteins and small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites.
• snRNPs are in the cell nucleus and are composed of several protein molecules and a small nuclear RNA molecule (snRNA).
• Each snRNA is about 150 nucleotides long.
• The spliceosome interacts with certain sites along an intron, releasing the introns and joining the two exons that flanked the introns.

Ribozymes

• These are RNA molecules which function as enzymes, and catalyze the breakage and formation of phosphodiester bonds during RNA splicing.
• snRNAs appear to play a major role in catalytic processes.
• The idea of a catalytic role for snRNA came from the discovery of ribozymes
• Ribozymes are RNA molecules that function as enzymes.
• The discovery of ribozymes made the statement "All biological catalysts are proteins" obsolete, as small nuclear RNA (snRNA) can also act as one.
• The single-stranded nature of RNA plays an important role in allowing certain RNA molecules to function as ribozymes.
• A region of the RNA molecule may base-pair with a complementary region elsewhere in the same molecule, creating a specific 3-D structure key to its ability to catalyze reactions.

Self-Splicing Introns

• In some organisms, splicing occurs without proteins or additional RNA molecules through self splicing introns.
• The intron RNA acts as a ribozyme and catalyzes its excision.
• For example, in the protozoan Tetrahymena, self-splicing occurs in the production of ribosomal RNA (rRNA).
• The pre-rRNA actually removes its own introns.

Introns and RNA Splicing

• These appear to have several functions.
• Some introns play a regulatory role in the cell, containing sequences that control gene activity such as enhancers or silencers.
• Splicing itself may regulate the passage of mRNA from the nucleus to the cytoplasm.
• Split genes enable one gene to encode for more than one polypeptide.

Alternative RNA Splicing

• This is a regulated process during gene expression resulting in a single gene coding for multiple proteins.
• Particular exons of a gene may be included within or excluded from the final, processed mRNA produced from that gene.
• Sex differences in fruit flies are from the splicing of RNA transcribed from certain genes.
• The Human Genome Project indicates that the phenomenon of alternative RNA splicing may explain why humans have a relatively small number of genes.
• Different exons code for different domains of a protein.
• Alternative splicing occurs as a normal phenomenon in eukaryotes
• It greatly increases the biodiversity of proteins that can be encoded by the genome.
• About 95% of multiexonic genes are alternatively spliced in humans.

Exon skipping in alternative RNA splicing

• A particular exon may be included in mRNAs under some conditions or in particular tissues and omitted from the mRNA in others.
• The production of alternatively spliced mRNAs is regulated by a system of trans-acting proteins, which bind to cis-acting sites on the primary transcript itself.
• Splicing activators promote the usage of a particular splice site, while splicing repressors reduce the usage of a particular site.

Calcitonin and CGRP

• An example of alternative splicing is the calcitonin or calcitonin gene-related peptide (CGRP).
• The primary RNA transcript undergoes tissue-specific alternative processing.
• Calcitonin mRNA is produced in thyroid C cells, and CGRP mRNA is produced in neurons of the central and peripheral nervous systems.

mRNA Processing

• All three of the mRNA-processing events (splicing, capping and polyadenylation) take place during transcription.
• Splicing begins when transcription is still underway.
• Capping occurs when nascent mRNA is about 30 nt long and the 5'-end of RNA first emerges from polymerase.
• Polyadenylation occurs when the still-growing mRNA is cut at the polyadenylation site.