Premed · Premed · General Biology 1
Lecture 21: Transcription
General Biology I — Molecular & Cellular
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the central dogma of molecular biology
- Explain the process of transcription in prokaryotes, including initiation, elongation, and termination
- Identify the roles of RNA polymerase, promoters, and terminators
- Describe eukaryotic transcription and the role of transcription factors
- Explain the three types of post-transcriptional RNA processing in eukaryotes (5' cap, 3' poly-A tail, splicing)
Lecture Content
I. The Central Dogma of Molecular Biology
Proposed by Francis Crick in 1958, the central dogma describes the flow of genetic information in cells: DNA -> RNA -> Protein. Transcription is the synthesis of RNA from a DNA template, and translation is the synthesis of a polypeptide directed by the RNA message. A few notable exceptions exist: reverse transcription (RNA -> DNA) occurs in retroviruses like HIV, which use the enzyme reverse transcriptase to convert their RNA genomes into DNA. Some RNA viruses replicate their genomes via RNA-dependent RNA replication (RNA -> RNA). And prions represent the remarkable case of protein influencing protein conformation without any nucleic acid template.
II. Overview of Transcription
Transcription is the process of synthesizing an RNA molecule using one strand of DNA as a template. It shares certain features with DNA replication--both are template-directed, involve complementary base pairing, and proceed in the 5' to 3' direction using nucleoside triphosphate substrates. However, there are critical differences. Transcription uses RNA polymerase rather than DNA polymerase, employs ribonucleotides (ATP, UTP, GTP, CTP) with uracil replacing thymine, does not require a primer, transcribes only one strand of the DNA for any given gene, and produces a single-stranded RNA product.
III. Key Terminology
The template strand (also called the antisense strand) is the DNA strand read by RNA polymerase in the 3' to 5' direction. The coding strand (sense strand or non-template strand) has the same sequence as the resulting mRNA (in the 5' to 3' direction), except with thymine instead of uracil. The promoter is the DNA sequence upstream of the gene that signals where transcription begins, while the terminator signals where it ends. Together, the stretch from promoter to terminator constitutes the transcription unit. The +1 site marks the transcription start site; positions upstream are numbered with negative values, positions downstream with positive values.
IV. Prokaryotic Transcription
Bacteria use a single RNA polymerase for all transcription. The core enzyme (composed of alpha2-beta-beta'-omega subunits) carries out RNA synthesis, but it cannot recognize promoters on its own. For that, it requires the sigma factor (sigma-70), which together with the core enzyme forms the holoenzyme.
A. Initiation
The sigma factor scans the DNA and recognizes two conserved promoter sequences: the -10 region (Pribnow box), with the consensus sequence TATAAT located approximately 10 base pairs upstream of the start site, and the -35 region, with the consensus TTGACA. RNA polymerase binds to the promoter, initially forming a closed complex in which the DNA remains double-stranded. The enzyme then melts (unwinds) approximately 12-14 base pairs to form an open complex, creating the transcription bubble. The first nucleoside triphosphate is placed opposite the +1 position on the template strand, and RNA synthesis begins without a primer. After about 8-10 nucleotides have been synthesized and the polymerase clears the promoter, the sigma factor is released.
B. Elongation
The core enzyme moves along the template strand in the 3' to 5' direction, synthesizing RNA 5' to 3' at a rate of approximately 40-80 nucleotides per second in E. coli. The transcription bubble (~12-14 base pairs of unwound DNA) travels with the enzyme. Behind the polymerase, the DNA rewinds into its double-helical form. Only about 8 base pairs of RNA-DNA hybrid exist at any time; the growing RNA strand peels away and exits through a channel in the enzyme. Multiple RNA polymerases can transcribe the same gene simultaneously, forming a "Christmas tree" pattern visible by electron microscopy and greatly increasing mRNA output.
C. Termination
Prokaryotic transcription terminates by one of two mechanisms. In rho-independent (intrinsic) termination, a GC-rich palindromic sequence in the newly synthesized RNA folds into a stable hairpin loop (stem-loop structure), followed by a run of uracil residues (transcribed from a run of adenines on the template). The weak rU-dA base pairs, combined with the destabilizing effect of the hairpin on the polymerase, cause the enzyme to dissociate and release the RNA. In rho-dependent termination, the rho protein--an ATP-dependent RNA helicase--binds to a rut site (rho utilization site) on the nascent RNA and translocates along it in the 5' to 3' direction. When rho catches up to a paused polymerase, it unwinds the RNA-DNA hybrid, releasing the transcript.
<image>A diagram of prokaryotic transcription. Top panel (Initiation): RNA polymerase holoenzyme (with sigma factor) binding to the promoter region (-35 and -10 boxes labeled). The DNA unwinds to form a transcription bubble. The template strand (3' to 5') and coding strand (5' to 3') are labeled. The +1 start site is marked. Middle panel (Elongation): The core enzyme moves along the template, with the growing RNA strand emerging from the polymerase. The transcription bubble, RNA-DNA hybrid region, and direction of movement are shown. Bottom panel (Termination): Two sub-panels — rho-independent termination showing a GC-rich hairpin loop in the RNA followed by a poly-U tail causing dissociation, and rho-dependent termination showing rho protein catching up to a paused polymerase.</image>
V. Eukaryotic Transcription
Eukaryotic transcription is substantially more complex than its prokaryotic counterpart. Three different RNA polymerases share the workload: RNA Polymerase I transcribes ribosomal RNA genes in the nucleolus, RNA Polymerase II transcribes messenger RNA (as well as some snRNAs and miRNAs), and RNA Polymerase III transcribes transfer RNA, 5S rRNA, and other small RNAs. The discussion here focuses on RNA Polymerase II and the synthesis of mRNA.
Eukaryotic promoters include several elements: the TATA box (consensus TATAAA, located ~25-30 bp upstream of the start site), the initiator (Inr) element surrounding the +1 site, CpG islands (common in mammalian promoters), and more distant regulatory elements such as enhancers (which increase transcription) and silencers (which decrease it). Unlike bacterial RNA polymerase, eukaryotic RNA Pol II cannot bind the promoter alone. It requires a set of general transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Assembly of the pre-initiation complex (PIC) begins when TFIID recognizes the TATA box through its TBP (TATA-binding protein) subunit. The remaining factors and RNA Pol II assemble in sequence. TFIIH plays a dual role: its helicase activity unwinds the DNA to form the transcription bubble, and its kinase activity phosphorylates the C-terminal domain (CTD) of RNA Pol II, triggering promoter clearance and the transition to productive elongation. The Mediator complex, a large multi-subunit assembly, bridges gene-specific transcription factors bound at enhancers with the general machinery at the promoter.
VI. Post-Transcriptional RNA Processing (Eukaryotes)
The primary transcript (pre-mRNA) produced by RNA Pol II must undergo three major processing steps in the nucleus before it is exported to the cytoplasm as mature mRNA.
A. 5' Capping
A modified guanine nucleotide--7-methylguanosine (m7G)--is added to the 5' end of the pre-mRNA via an unusual 5'-5' triphosphate linkage. This cap is added co-transcriptionally (while transcription is still in progress) and serves multiple functions: it protects the mRNA from degradation by exonucleases, is required for ribosome recognition during translation initiation, and facilitates nuclear export.
B. 3' Polyadenylation
A signal sequence (AAUAAA) near the 3' end of the pre-mRNA is recognized by cleavage and polyadenylation factors. The pre-mRNA is cleaved 10-35 nucleotides downstream of this signal, and poly-A polymerase adds a tail of approximately 100-250 adenine nucleotides (the poly-A tail). The poly-A tail protects the mRNA from degradation, aids in nuclear export, and is required for efficient translation initiation. The length of the poly-A tail can serve as a regulatory signal: shortening (deadenylation) is often the first step in mRNA decay.
C. RNA Splicing
Eukaryotic genes are typically interrupted by introns (intervening, non-coding sequences) that must be removed, and the remaining exons (expressed sequences) are joined together to form the continuous coding sequence. This process, called splicing, is carried out by the spliceosome, a massive molecular machine composed of five small nuclear ribonucleoproteins (snRNPs: U1, U2, U4, U5, and U6) and numerous associated proteins. The spliceosome recognizes conserved sequences at intron boundaries: a GU at the 5' splice site, an AG at the 3' splice site, and a branch-point adenine within the intron. Splicing proceeds through two sequential transesterification reactions: first, the 2'-OH of the branch-point adenine attacks the 5' splice site, forming a lariat intermediate; then, the free 3'-OH of the upstream exon attacks the 3' splice site, joining the exons and releasing the intron as a lariat that is subsequently degraded.
Alternative splicing allows a single pre-mRNA to be spliced in different ways, producing different mature mRNAs and therefore different proteins from the same gene. This is a major source of protein diversity in eukaryotes--the human genome contains approximately 20,000 genes but produces over 100,000 distinct proteins, with an estimated 95% of multi-exon genes undergoing alternative splicing. The Drosophila Dscam gene can theoretically generate over 38,000 mRNA variants, making it one of the most extreme examples of this phenomenon.
<image>A three-panel diagram of eukaryotic mRNA processing. Panel A: 5' capping — the 5' end of the growing pre-mRNA receives a 7-methylguanosine cap attached via a 5'-5' triphosphate bridge. Panel B: 3' polyadenylation — the AAUAAA signal is recognized, the pre-mRNA is cleaved, and poly-A polymerase adds ~200 A residues. Panel C: RNA splicing — a pre-mRNA with three exons and two introns is shown. The spliceosome (shown as a large complex of snRNPs — U1 at the 5' splice site, U2 at the branch point) assembles on the intron. Two transesterification steps are shown: lariat formation and exon ligation. The final mature mRNA has exons joined together, with the 5' cap and poly-A tail. An inset shows alternative splicing: the same pre-mRNA can be spliced to include or skip certain exons, producing different mRNA variants.</image>
