Gene Expression Flashcards

Learning Objectives

• Define and explain the “central dogma of life.”

• Outline the process of genetic transcription in eukaryotes.

• Summarize the process of genetic translation.

• Decode the sequence of an mRNA into the amino acid sequence of a protein.

• Understand the impact of various DNA mutations on the functioning of organisms.

The Central Dogma of Life

• Information flow in all organisms takes place from DNA to RNA to protein.

  • DNA is transcribed to RNA via complementary base pairing rules (with U instead of T in the transcript).

  • The RNA transcript, specifically pre-mRNA, is then processed into mRNA.

  • The mature mRNA is then translated to an amino acid polypeptide by ribosomes in the cytoplasm.

  • Final folding and modifications of the polypeptide (Golgi apparatus) lead to functional proteins that perform the work happening in cells.

Regulating Gene Expression

• All cells control or regulate the synthesis of proteins from information encoded in their DNA.

• Gene expression is the process of turning a gene “ON” to produce RNA and protein.

• Gene regulation is how a cell controls which genes, out of the many genes in its genome, are expressed (“turned on”).

• Each cell type in the body has a different set of active genes, despite almost all cells containing the exact same DNA, thanks to gene regulation.

• The regulation of gene expression conserves energy and space.

Transcription and Translation

• Transcription takes place in the nucleus, using DNA as a template to make an RNA (mRNA) molecule.

• During transcription, one strand of DNA is used as the template by the enzyme RNA polymerase to produce an mRNA molecule with complementary base pairs (A à U; T à A; G à C; C à G).

• In translation, triplets, called codons, of mRNA are read by ribosomes.

• These codons inform the ribosomes which amino acids to bind to each other.

Steps of Transcription

• Initiation:

  • Occurs when the enzyme RNA polymerase binds to a region of a gene called the promoter.

  • This signals the DNA to unwind so the enzyme can ‘‘read’’ the bases in one of the DNA strands.

  • Eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase (unlike prokaryotes).

• Elongation:

  • Addition of nucleotides to the mRNA strand.

  • RNA polymerase reads the unwound DNA strand and builds the mRNA molecule, using complementary base pairs.

  • There is a brief time during this process when the newly formed RNA is bound to the unwound DNA.

  • During this process, an adenine (A) in the DNA binds to a uracil (U) in the RNA.

• Termination:

  • The ending of transcription and occurs when RNA polymerase crosses a termination sequence in the gene.

  • The mRNA strand is complete and detaches from DNA.

Structure of a Eukaryotic RNA Polymerase II Promoter

• Transcription factors recognize the TATAA box in the promoter.

• RNA polymerase then binds and forms the transcription initiation complex.

RNA Processing: From pre-mRNA to mRNA

• After transcription by RNA Pol II, eukaryotic pre-mRNAs must undergo several processing steps before they can be translated.

• The eukaryotic pre-mRNA undergoes extensive processing before it is ready to be translated.

• The additional steps involved in eukaryotic mRNA maturation create a molecule with a much longer half-life than a prokaryotic mRNA.

• Eukaryotic (and prokaryotic) tRNAs and rRNAs also undergo processing before they can function as components in the protein synthesis machinery.

RNA Processing

• RNA polymerase II is in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs.

• Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation:

  • Introns must be spliced out.

  • A 5′ cap and 3′ poly-A tail are added.

• Splicing has an evolutionary function to create much more proteins than there are genes in a genome!

• Adding the 5’cap and 3’ poly-A-tail stabilizes the fragile mRNA and helps with ribosome recognition!

Pre-mRNA Splicing

• Pre-mRNA splicing involves the precise removal of introns from the primary RNA transcript.

• The splicing process is catalyzed by protein complexes called spliceosomes that are composed of proteins and RNA molecules called snRNAs.

• Spliceosomes recognize sequences at the 5′ and 3′ end of the intron.

RNA Polymerase I and III

• RNA polymerase I

  • Located in the nucleolus, a specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes.

  • The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein.

• RNA polymerase III

  • Also located in the nucleus.

  • This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs.

Translation

• Translation, or protein synthesis is the decoding of an mRNA message into a polypeptide product.

• A peptide bond links the carboxyl end of one amino acid with the amino end of another, expelling one water molecule.

Translation Takes Place in the Cytoplasm

• Ribosomes use the mRNA as a template to make a polypeptide molecule.

• During translation, the ribosomes bind tRNA molecules.

• With their anticodons, these tRNA molecules bind to the codons on the mRNA.

• At the same time, they carry the correct amino acid on its other side.

• The amino acid they carry corresponds to their anticodon (according to the codon table).

• The tRNA thus functions as an adapter between the nucleotide language of mRNA and the amino acid language of proteins.

Ribosome RNA Translation

• A ribosome is a complex macromolecule composed of structural and catalytic rRNAs and many distinct polypeptides.

• In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.

• Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and rough endoplasmic reticulum in eukaryotes.

• Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the ribosomes just outside their outer membranes in the cytoplasm.

Steps of Translation

• Initiation:

  • Translation begins when an initiator tRNA anticodon recognizes a codon on mRNA.

  • The large ribosomal subunit joins the small subunit, and a second tRNA is recruited.

• Elongation:

  • As the mRNA moves relative to the ribosome, the polypeptide chain is formed.

• Termination:

  • Entry of a protein release factor into the A site terminates translation, and the components dissociate.

  • A polypeptide is ‘born’.

Codons

• Amino acids are encoded by nucleotide triplets.

• A codon is a triplet that specifies a particular amino acid or stop codon.

• A given amino acid can be encoded by more than one nucleotide triplet.

• This is often referred to by saying the genetic code is degenerate.

The Genetic Code

• This figure shows the genetic code for translating each nucleotide triplet in mRNA into an amino acid or a termination signal in a nascent protein.

Protein Modifications

• During and after translation, individual amino acids may be chemically modified, signal sequences may be appended, and the new protein “folds” into a distinct three-dimensional structure as a result of intramolecular interactions.

• A signal sequence is a short tail of amino acids that directs a protein to a specific cellular compartment.

• Many proteins fold spontaneously, but some proteins require helper molecules, called chaperones, to prevent them from aggregating during the complicated process of folding.

• Even if a protein is properly specified by its corresponding mRNA, it could take on a completely dysfunctional shape due to environmental conditions at this stage.

DNA Mutations

• A mutation is a change that occurs in a DNA sequence, either due to mistakes when the DNA is copied or as the result of environmental factors such as UV light and cigarette smoke.

• Often cells can recognize any potentially mutation-causing damage and repair it before it becomes a fixed mutation.

• Mutations contribute to genetic variation within species.

• Mutations can also be inherited, particularly if they have a positive effect.

• However, mutation can also disrupt normal gene activity and cause diseases, like cancer.

• There are non-sense, missense, silent, or frameshift mutations.

• The deletion of one or two nucleotides from a gene shifts the reading frame of an mRNA and changes the entire protein sequence, creating a nonfunctional protein or terminating protein synthesis altogether.

Gene Mutations

• Gene mutations can be classified in two major ways:

  • Hereditary mutations are inherited from a parent and are present throughout a person’s life in virtually every cell in the body. When an egg and a sperm cell unite, the resulting fertilized egg cell receives DNA from both parents. If this DNA has a mutation, the child that grows from the fertilized egg will have the mutation in each of his or her cells.

  • Acquired (or somatic) mutations occur at some time during a person’s life and are present only in certain cells, not in every cell in the body. These changes can be caused by environmental factors or can occur if a mistake is made as DNA copies itself during cell division. Acquired mutations in somatic cells (cells other than sperm and egg cells) cannot be passed on to the next generation.

Effect of Mutations on Primary Structure of a Protein

• The unique sequence for every protein is ultimately determined by the nucleic acid sequence of the gene encoding the protein.

• A change in the nucleotide sequence of the gene’s coding region (mutation) may lead to a different amino acid being added to the growing polypeptide chain, causing a change in protein structure and function.

Mosaicism

• Somatic mutations that happen in a single cell early in embryonic development can lead to a situation called mosaicism.

• These genetic changes are not present in a parent’s egg or sperm cells, or in the fertilized egg, but happen a bit later when the embryo includes several cells.

• As all the cells divide during growth and development, cells that arise from the cell with the altered gene will have the mutation, while other cells will not.

• Depending on the mutation and how many cells are affected, mosaicism may or may not cause health problems.

Polymorphism

• Genetic alterations that occur in more than 1 percent of the population are called polymorphisms.

• They are common enough to be considered a normal variation in the DNA.

• Polymorphisms are responsible for many of the normal differences between people such as eye color, hair color, and blood type.

• Although many polymorphisms have no negative effects on a person’s health, some of these variations may influence the risk of developing certain disorders.

Practice Question #1

• Match the process with the summary:

  • RNA to Protein - Translation

  • DNA to DNA - Replication

  • DNA to RNA - Transcription

Practice Question #2

• Genetic engineering often uses bacteria to synthesize proteins for use in humans. How is this possible, and why might bacteria be better for this purpose than eukaryotic cells?

Quick Review

• Outline the process of eukaryotic transcription.

• Summarize the process of translation.

• Be able to decode the sequence of an mRNA into the amino acid sequence of a protein.

• Identify the central dogma of life.

Transcription and translation are fundamental processes involved in gene expression, wherein the information encoded in genes is utilized to synthesize proteins.

Transcription

Transcription is the first step in the gene expression process, during which a specific segment of DNA is copied into RNA. This process occurs in the nucleus of eukaryotic cells and involves several key steps:

1. Initiation:

   • The enzyme RNA polymerase binds to a specific region of the gene called the promoter.

   • This binding triggers the unwinding of the DNA, allowing RNA polymerase to access the template strand.

   • Eukaryotes require additional proteins known as transcription factors to bind to the promoter region before RNA polymerase can initiate transcription.

2. Elongation:

   • RNA polymerase reads the unwound DNA template strand and begins synthesizing the RNA molecule by adding complementary RNA nucleotides. For example, adenine (A) on the DNA pairs with uracil (U) in the RNA, thymine (T) pairs with adenine (A), guanine (G) pairs with cytosine (C), and cytosine (C) pairs with guanine (G).

   • The RNA molecule elongates as more nucleotides are added, creating a primary RNA transcript (pre-mRNA).

3. Termination:

   • Transcription concludes when RNA polymerase reaches a termination sequence in the DNA. Upon reaching this sequence, RNA polymerase detaches from the DNA, releasing the newly formed RNA strand.

RNA Processing (in Eukaryotes)

In eukaryotic organisms, pre-mRNA undergoes a series of modifications before being translated into protein:

• Splicing: Introns (non-coding sequences) are removed from the pre-mRNA, while exons (coding sequences) are joined together. This process occurs via spliceosomes, which are composed of proteins and small nuclear RNAs (snRNAs).

• 5' Capping: A 5' cap is added to the beginning of the mRNA molecule to protect it from degradation and assist in ribosome recognition during translation.

• Polyadenylation: A poly-A tail (a series of adenine nucleotides) is added to the 3' end of the mRNA, enhancing its stability and export from the nucleus.

Once these modifications are complete, the mature mRNA is transported from the nucleus to the cytoplasm where translation occurs.

Translation

Translation is the process through which the sequence of nucleotides in mRNA is decoded to produce a polypeptide (protein). This process takes place in the cytoplasm and involves multiple components, including ribosomes, transfer RNAs (tRNAs), and the mRNA itself:

1. Initiation:

   • Translation begins when an initiator tRNA, carrying the first amino acid (usually methionine), binds to the start codon (AUG) on the mRNA.

   • The small ribosomal subunit associates with the mRNA and the initiator tRNA. The large ribosomal subunit then joins to form a complete ribosome.

2. Elongation:

   • As the ribosome moves along the mRNA, tRNA molecules, with their corresponding amino acids and anticodons, sequentially bind to the codons on the mRNA.

   • The ribosome forms peptide bonds between amino acids, creating a growing polypeptide chain. Each tRNA leaves after transferring its amino acid, allowing for the next tRNA to bind.

3. Termination:

   • Translation ends when a stop codon (UAA, UAG, UGA) is encountered. A protein release factor enters the ribosome, prompting the release of the newly synthesized polypeptide.

   • The ribosomal subunits disassemble, and the mRNA is freed to be used again or degraded.

Conclusion

Transcription and translation together represent the central dogma of molecular biology, where genetic information flows from DNA to RNA to protein. Understanding these processes is crucial for grasping how cells function and respond to various stimuli, adapt their functions, and maintain homeostasis. The intricacies of transcription and translation highlight the precision of cellular mechanisms that underpin gene expression.