BIOB11 LECTURE 5
RNA & Transcription Learning Objectives
Explain how moving from information storage (DNA) to information use (RNA and proteins) includes amplification:
Amplification occurs because one DNA sequence can be transcribed into many mRNA copies. Each mRNA can then be translated into many protein molecules, leading to a significant increase in the number of functional molecules (proteins) compared to the original DNA template.
Identify differences & similarities between the structures and functions of RNA vs DNA:
Similarities:
Both are nucleic acids with a sugar-phosphate backbone and nitrogenous bases.
Both have 5’-3’ directionality.
Both utilize complementary base-pairing.
Differences:
RNA is single-stranded, while DNA is double-stranded.
RNA has ribose sugar, while DNA has deoxyribose sugar.
RNA uses uracil (U) instead of thymine (T), which is found in DNA.
DNA is used for long-term information storage; RNA is for short-term information use (mRNA), structural (rRNA), and functional (tRNA, catalytic RNA).
Explain the general steps in bacterial transcription, including how the template strand & directionality of DNA is selected and mechanisms involved in initiation and termination:
Initiation: RNA polymerase binds to the promoter region on the DNA with the help of sigma factor (\sigma), which increases its affinity for promoter sequences. The promoter's orientation determines the direction of transcription.
Elongation: RNA polymerase opens up the DNA strands and synthesizes a complementary RNA strand using ribonucleoside triphosphates (rNTPs). RNA synthesis occurs in the 5’ to 3’ direction, reading the template strand in the 3’ to 5’ direction.
Termination: Transcription ends when the RNA polymerase reaches a specific termination sequence on the DNA. This can be intrinsic, involving stem-loop hairpins that cause the RNA polymerase to pause, or protein-mediated, involving proteins like rho factor (\rho).
Identify the 5’ to 3’ ends of the RNA (and template/coding DNA strands) during transcription and be able to predict RNA sequences from DNA sequences:
During transcription, the RNA strand is synthesized in the 5’ to 3’ direction, using the template strand of DNA, which runs 3’ to 5’. The coding strand (non-template strand) has the same sequence as the RNA, except T is replaced by U.
To predict the RNA sequence from a given DNA template sequence, remember to use the complementary base pairs (A with U, G with C) and maintain the 5’ to 3’ direction.
Explain what a consensus sequence is and how this might be determined by researchers:
A consensus sequence is an idealized sequence showing the most frequently occurring bases at each position in a set of related sequences. Researchers determine it by aligning multiple similar sequences and identifying the most common nucleotide at each position.
Identify differences between eukaryotic and prokaryotic transcription including why they can differ with respect to the ratios of promoters to genes to potential types of proteins produced:
Eukaryotic Transcription:
Occurs in the nucleus.
Involves RNA processing (5’ capping, splicing, 3’ polyadenylation).
Uses monocistronic mRNA (one promoter per gene).
Prokaryotic Transcription:
Occurs in the cytoplasm.
Transcription and translation are coupled.
Uses polycistronic mRNA (one promoter for multiple genes).
Eukaryotes can produce a greater variety of proteins due to alternative splicing and the fact that each gene has its own promoter, allowing for finer control.
Explain what alternative splicing is and how it could impact cell function:
Alternative splicing is a process where different combinations of exons from the same gene are joined together, resulting in multiple different mRNA transcripts. This allows one gene to code for multiple proteins (splice isoforms) with different functions, thus increasing the diversity of proteins that can be produced from a single gene. It can impact cell function by producing proteins with tissue-specific or condition-specific activities.
Describe the major types of RNA in eukaryotes and which RNA polymerase synthesizes each:
mRNA (messenger RNA): carries the coding sequence for protein synthesis (RNA Polymerase II).
rRNA (ribosomal RNA): forms the basic structure of the ribosome and catalyzes protein synthesis (RNA Polymerase I and III).
tRNA (transfer RNA): central to protein synthesis as adaptors between mRNA and amino acids (RNA Polymerase III).
snRNA (small nuclear RNA): functions in a variety of nuclear processes, including the splicing of pre-mRNA (RNA Polymerase II and III).
snoRNA (small nucleolar RNA): help to process and chemically modify rRNAs (RNA Polymerase II).
Describe roles of each of the following in eukaryotic mRNA transcription and RNA processing: TATA box, GTFs (TFIID, TBP, TFIIH), transcription factors, RNA pol II, chromatin remodeling factors and transcription elongation factors, capping enzyme, RNA methyltransferase, CPSF, CstF, Poly (A) Polymerase:
TATA box: A promoter sequence that indicates where a genetic sequence can be read and decoded. It is the site of assembly of the preinitiation complex (PIC).
GTFs (General Transcription Factors):
TFIID: Recognizes the TATA box via its subunit TBP (TATA-binding protein).
TBP (TATA-binding protein): Binds to the TATA box and bends the DNA, serving as a platform for the assembly of other transcription factors.
TFIIH: Unwinds DNA at the transcription start point and phosphorylates the RNA polymerase II CTD to initiate elongation.
RNA pol II: Synthesizes mRNA precursors (pre-mRNA).
Chromatin remodeling factors: Facilitate transcription by altering chromatin structure, making DNA more accessible to RNA polymerase II.
Transcription elongation factors: Help the polymerase transcribe through nucleosomes.
Capping enzyme: Adds the 5’ methylguanosine cap to the mRNA.
RNA methyltransferase: Methylates the guanine base and the ribose to which the GMP was attached during capping.
CPSF (Cleavage and Polyadenylation Specificity Factor): Binds to the AAUAAA signal sequence and is involved in cleaving the RNA transcript.
CstF (Cleavage Stimulation Factor): Also binds to the AAUAAA signal sequence and is involved in cleaving the RNA transcript.
Poly (A) Polymerase: Adds the poly(A) tail to the 3’ end of the mRNA.
Identify functions for the RNA polymerase CTD and the role of CTD phosphorylation:
The CTD (C-terminal domain) of RNA polymerase II plays a crucial role in coordinating mRNA synthesis and processing. It serves as a binding site for various proteins involved in capping, splicing, and polyadenylation.
CTD phosphorylation by TFIIH signals the start of elongation, recruits processing proteins, and coordinates the different stages of mRNA processing.
Draw a primary transcript of pre-mRNA and a processed mRNA and label the introns vs exons, coding regions, 5’ cap, 3’ poly(A) tail, 3’ UTR, and 5’ UTR:
(Unfortunately, I am unable to draw images. However, descriptions of each element have been provided below.)
Primary transcript (pre-mRNA): Contains introns, exons, 5’ UTR, 3’ UTR, but lacks a 5’ cap and 3’ poly(A) tail.
Introns: Non-coding regions that are spliced out.
Exons: Coding regions that will be included in the mature mRNA.
5’ UTR (untranslated region): Region at the 5’ end that is not translated into protein.
3’ UTR (untranslated region): Region at the 3’ end that is not translated into protein.
Processed mRNA: Lacks introns, and includes a 5’ cap and 3’ poly(A) tail.
5’ cap: A modified guanine nucleotide added to the 5’ end.
3’ poly(A) tail: A string of adenine nucleotides added to the 3’ end.
Identify functions for the 5’ cap and 3’ poly(A) tail and describe how/when they are added:
5’ cap:
Function: Protects the mRNA from degradation by exonucleases, helps in transport out of the nucleus, and promotes translation initiation.
Addition: Added to the 5’ end of the mRNA shortly after transcription initiation.
3’ poly(A) tail:
Function: Protects the mRNA from degradation, enhances translation, and aids in the export of mRNA from the nucleus.
Addition: Added to the 3’ end of the mRNA after cleavage downstream of a