MCAT Biochemistry Review
Chapter 7: RNA and the Genetic Code
Although DNA contains the actual coding sequence for a protein, the machinery to generate that protein is located in the cytoplasm. DNA cannot leave the nucleus, as it will be quickly degraded, so it must use RNA to transmit genetic information. The creation of mRNA from a DNA template is known as transcription, and while mRNA is the only type of RNA that carries information from DNA directly, there are many other types of RNA that exist, two of which will play important roles during protein translation: transfer RNA (tRNA) and ribosomal RNA (rRNA).
When we transcribe information, we use the same language to write it down (like in court, when the judge asks the court reporter to read back the transcript—the reporter speaks the same language as written). Translation is exactly what it says: we are changing the language. RNA translation changes the language from nucleotides to amino acids.
MECHANISM OF TRANSCRIPTION
Transcription produces a copy of only one of the two strands of DNA. During initiation of transcription, several enzymes, including helicase and topoisomerase, are involved in unwinding the double-stranded DNA and preventing formation of supercoils, as described in Chapter 6 of MCAT Biochemistry Review. This step is important in allowing the transcriptional machinery access to the DNA and the particular gene of interest. Transcription results in a single strand of mRNA, synthesized from one of the two nucleotide strands of DNA called the template strand (or theantisense strand). The newly synthesized mRNA strand is both antiparallel and complementary to the DNA template strand.
RNA is synthesized by a DNA-dependent RNA polymerase; RNA polymerase locates genes by searching for specialized DNA regions known as promoters. In eukaryotes, RNA polymerase II is the main player in transcribing mRNA, and its binding site in the promoter region is known as the TATA box, named for its high concentration of thymine and adenine bases. Transcription factors help the RNA polymerase locate and bind to this promoter region of the DNA, helping to establish where transcription will start. Unlike DNA polymerase III, which we reviewed during DNA replication, RNA polymerase does not require an RNA primer to start generating a transcript.
In eukaryotes, there are three types of RNA polymerases, but only one is involved in the transcription of mRNA:
· RNA polymerase I is located in the nucleolus and synthesizes rRNA
· RNA polymerase II is located in the nucleus and synthesizes hnRNA (pre-processed mRNA) and some small nuclear RNA (snRNA)
· RNA polymerase III is located in the nucleus and synthesizes tRNA and some rRNA
RNA polymerase travels along the template strand in the 3′ → 5′ direction, which allows for the construction of transcribed mRNA in the 5′ → 3′ direction. Unlike DNA polymerase, RNA polymerase does not proofread its work, so the synthesized transcript will not be edited. The codingstrand (or sense strand) of DNA is not used as a template during transcription. Because the coding strand is also complementary to the template strand, it is identical to the mRNA transcript except that all the thymine nucleotides in DNA have been replaced with uracil in the mRNA molecule.
Transcription is subject to the 5′ → 3′ rule, just like DNA synthesis. Synthesis of nucleic acids always occurs in the 5′ → 3′ direction.
In the vicinity of a gene, a numbering system is used to identify the location of important bases in the DNA strand, as shown in Figure 7.8. The first base transcribed from DNA to RNA is defined as the +1 base of that gene region. Bases to the left of this start point (upstream, or toward the 5′ end) are given negative numbers: –1, –2, –3, and so on. Bases to the right (downstream, or toward the 3′ end) are denoted with positive numbers: +2, +3, +4, and so on. Thus, no nucleotide in the gene is numbered 0. The TATA box, where RNA polymerase II binds, usually falls around –25.
Transcription will continue along the DNA coding region until the RNA polymerase reaches a termination sequence or stop signal, which results in the termination of transcription. The DNA double helix then reforms, and the primary transcript formed is termed heterogeneous nuclear RNA (hnRNA). mRNA is derived from hnRNA via posttranscriptional modifications, as described below.
Figure 7.8. Transcription of DNA to hnRNA
Before the hnRNA can leave the nucleus and be translated to protein, it must undergo three specific processes to allow it to interact with the ribosome and survive the conditions of the cytoplasm, as demonstrated in Figure 7.9. You can think of the nucleus as the happy home of the cell; the DNA strands are the parents, and the hnRNA is their child. The child must mature if he or she is to survive.
Figure 7.9. Processing Eukaryotic hnRNA to Form mRNA
The MCAT commonly tests post-transcriptional processing:
· Intron/exon splicing
· 5′ cap
· 3′ poly-A tail
Splicing: Introns and Exons
Maturation of the hnRNA includes splicing of the transcript to remove noncoding sequences (introns) and ligate coding sequences (exons) together. Splicing is accomplished by the spliceosome. In the spliceosome, small nuclear RNA (snRNA) molecules couple with proteins known assmall nuclear ribonucleoproteins (also known as snRNPs, or “snurps”). The snRNP/snRNA complex recognizes both the 5′ and 3′ splice sites of the introns. These noncoding sequences are excised in the form of a lariat (lasso-shaped structure) and then degraded.
The evolutionary function of introns in eukaryotic cells is not currently well-understood; however, scientists hypothesize that introns play an important role in the regulation of cellular gene expression levels and in maintaining the size of our genome. The existence of introns has also been hypothesized to allow for rapid protein evolution. Many eukaryotic proteins share peptide sequences in common, suggesting that the genes encoding for these particular peptides may employ a modular function; that is, they contain standard sequences that can be swapped in and out, depending on the needs of the cell.
At the 5′ end of the hnRNA molecule, a 7-methylguanylate triphosphate cap is added. The cap is actually added during the process of transcription and is recognized by the ribosome as the binding site. It also protects the mRNA from degradation in the cytoplasm.
3′ Poly-A Tail
A polyadenosyl (poly-A) tail is added to the 3′ end of the mRNA transcript and protects the message against rapid degradation. It is composed of adenine bases. Think of the poly-A tail as a fuse for a “time bomb” for the mRNA transcript: as soon as the mRNA leaves the nucleus, it will start to get degraded from its 3′ end. The longer the poly-A tail, the more time the mRNA will be able to survive before being digested in the cytoplasm. The poly-A tail also assists with export of the mature mRNA from the nucleus.
Introns stay in the nucleus; exons will exit the nucleus as part of the mRNA.
At this point, when only the exons remain and the cap and tail have been added, the cell has created the mature mRNA that can now be transported into the cytoplasm for protein translation. Untranslated regions of the mRNA (UTRs) will still exist at the 5′ and 3′ edges of the transcript because the ribosome initiates translation at the start codon (AUG) and will end at a stop codon (UAA, UGA, UAG).
For some genes in eukaryotic cells, however, the primary transcript of hnRNA may be spliced together in different ways to produce multiple variants of proteins encoded by the same original gene. This process is known as alternative splicing, and it is illustrated in Figure 7.10. By utilizing alternative splicing, an organism can make many more different proteins from a limited number of genes. For reference, humans are estimated to make at least 100,000 proteins, but the number of human genes is only about 20,000–25,000. Don't worry about memorizing these numbers, though; they are constantly changing with new research. Alternative splicing is also known to function in the regulation of gene expression, in addition to generating protein diversity.
Figure 7.10. Alternative Splicing of Eukaryotic hnRNA to Produce Different Proteins
Mutations in splice sites can lead to abnormal proteins. For example, mutations that interfere with proper splicing of β-globulin mRNA are responsible for some cases of β-thalassemia, a group of blood disorders that hinder the production and efficacy of hemoglobin in the blood. Splice site mutations are one of the few mutations in noncoding DNA that may still have an effect on the translated protein.
MCAT Concept Check 7.2:
Before you move on, assess your understanding of the material with these questions.
1. What is the role of each eukaryotic RNA polymerase?
· RNA polymerase I:
· RNA polymerase II:
· RNA polymerase III:
2. When starting transcription, where does RNA polymerase bind?
3. What are the three major posttranscriptional modifications that turn hnRNA into mature mRNA?
4. What is alternative splicing, and what does it accomplish?