401G
Lecture BCH 37
Andres
Lecture Summary: Regulation of transcription. General mechanisms-what are the major regulatory points? RNA processing: Capping, polyadenylation, splicing. Why process mammalian RNA? What role do SNrNPs play? Know structural details and consensus sequences.
Regulation of Prokaryotic Transcription: What are genes? As we defined previously, a gene is any DNA sequence that is transcribed into RNA. Why does a cell need to make these RNAs? Cell needs mRNA because they serve as the templates for protein synthesis. Cell needs rRNA and tRNA's because they are critically involved in protein synthesis. Cell needs all of the RNAs they make. However, a cell needs different amounts of these RNA molecules. How are RNA levels regulated? Primarily regulated by controlling how frequently each gene is transcribed to RNA. Initiation of Transcription is the major control point: If transcription of a certain gene initiates often, then a large number of transcripts are made from that gene. If transcription initiates very rarely, then few transcripts from that gene will be found in the cell from that particular gene.
So the central question is: What determines the frequency at which a specific gene is transcribed? The transcription rate is controlled by proteins that bind to specific DNA sequences in the promoters of a particular gene. Promoter sequences are usually found at the 5' end of a gene relative to the coding strand. Proteins are called "Transcription Factors". Each recognizes a particular DNA sequence. The binding of a transcription factor to a promoter sequence determines whether or not RNA polymerase initiates transcription of a particular gene. Both positive and Negative Mechanisms used to Regulate Transcription. Positive transcription factors act to increase transcription. Called Activators. Negative transcription factors act to decrease transcription. Called Repressors.
In prokaryotes regulation of gene expressed is controlled predominantly at the level of RNA transcription. Control occurs in two ways: 1) Induction: Genes are turned on in order to utilize an available substrate. 2) Repressed: Genes are turned off - often used when the enzymes are involved in a anabolic pathway. The end product of the pathway when in high cellular levels causes its own synthesis to be shut off by inhibiting further expression of necessary biosynthetic enzymes.
RNA processing. We have discussed the action of RNA polymerase and how it can make a single-stranded RNA copy, or transcript, of a gene. RNA transcription begins at the +1 Transcription Start site and ends at the transcriptional termination site. The RNA molecule therefore looks like this: 5'----------------------------3' However, in eukaryotes, most of these initial RNA transcripts are not synthesized in their final (functional) form. Cells must alter the original RNA transcript to make them functional. This process is called "RNA Processing". We will talk about two major kinds of RNA processing which occur in eukaryotes:
1.
Adding nucleotides at the 5' and 3' ends of RNA transcripts.
2. Specific Sequences of RNA Transcripts are removed. The remaining pieces are rejoined to form a smaller RNA molecule. These types of processing primarily involve messenger RNA. Extensive RNA processing is generally associated with increased RNA stability. This is particularly true for mRNA. Prokaryotic mRNA has a half-life of minutes while eukaryotic mRNA has a half-life of hours (tRNA and rRNA has a half-life of hours to days).
1). Processing of Messenger RNA at the 5' and 3' ends. A. Processing the 5' end of mRNA. In Eukaryotes, as in prokaryotes, transcription usually begins with an A or G. However, the free 5' phosphate is quickly modified. A phosphate is released by hydrolysis and then the newly made diphosphate attacks an alpha phosphate on a GTP to form an unusual 5' to 5' Cap.
The N-7 nitrogen is then methylated to form Cap 0. The adjacent riboses are then methylated in the 2' position to form cap1 and cap2. This modification does not occur on rRNAs or tRNAs.
Why do mRNAs have 5' Cap structures? 1. Cells have enzymes called 5' exonucleases that cleave nucleotides from the 5' ends of RNAs. The 5' Cap prevents these enzymes from cleaving the 5' end of mRNAs. This makes the mRNAs more stable. 2. 5' Cap serves as attachment site for mRNA to ribosomes to initiate translation. This the makes mRNA a better template for new protein synthesis.
B. Processing at the 3' end of mRNAs. At 3' end of eukaryotic mRNA transcripts contain a sequence that has the consensus sequence: 5' AAUAAA 3'. This sequence is called the "Polyadenylation Signal Sequence." This sequence motif is bound by a protein called the "Cleavage and Polyadenylation Specificity Factor" or "CPSF". CPSF interacts with an endonuclease which cuts the RNA transcript 10-20 nucleotides after the 5' AAUAAA 3' sequence. CPSF also interacts with a protein called "Poly A Polymerase" which adds many adenosine nucleotides to the 3' end of mRNA transcript after it has been cleaved. This process is called "Polyadenylation". The sequence of adenosine nucleotides on the 3' end of mRNAs is called a "Poly A Tail". Can be up to 250 nucleotides in length (actual length varies for different mRNAs).
The function of the 3' Poly A Tail is similar to the 5' Cap structure: 1). Protects mRNA from enzymes (3' exonucleases) which cleave the 3' ends of RNAs. A longer poly A tail gives a mRNA a longer life within the cell. 2). The A Tail also makes the mRNA a better template for protein synthesis. Two interesting properties of Poly A Polymerase: 1. Adenosine nucleotides are added one at a time. The polymerase dissociates and rebinds after each addition. The DNA and RNA polymerases we have talked about incorporate many nucleotides before dissociating. 2. No template sequence is required for the addition of adenine nucleotides. Remember: Both RNA and DNA polymerases require a template strand. 3. Adenine nucleotides are the only bases that can be added.
To summarize, Capping at the 5' end and Polyadenylation at the 3' end gives a mRNA that looks like this: 5' Cap----------------------------------------------AAAAAAAAAAAAA 3'
Second type of mRNA processing in eukaryotes. "Splicing." Most eukaryotic mRNAs are transcribed with internal sequences that must be removed before the mRNA can be used for protein synthesis. These internal sequences which must be removed are called "Introns". The coding portions of the mRNA (that remain after removal of the introns) are called "Exons". Intron= intervening sequence, which is removed. Exon= expressed information, which is retained in the mature mRNA.
Introns are removed, and the exons have to be joined: This process is called "Splicing".
Splicing is performed by an enzyme complex that contains both RNA and protein subunits: Called "small nuclear ribonucleoproteins" or "snRNPs". snRNPs recognize and bind ...