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Department of Biological and Environmental Sciences

Cell & Molecular Biology
Dr. David A. Johnson
Biol 405    4 Credits   Spring 2017  MWF 11:45-12:50 AM   PH

RNA Synthesis (Transcription) and RNA Processing
Text Ch 8: 259-263, 265-271, 295-310

[α is alpha, β
is beta, ω is omega, σ is sigma, ρ is rho]

DNA's role as the genetic material includes 1) carrying information (in its base sequence), 2) copying that information (replication), and 3) giving meaning to that information (determining traits). Genes accomplish their job in this last role by directing the activity of the cell, primarily by determining which proteins (including the all-important enzymes) the cell makes. By determining what enzymes the cell makes, the DNA controls all of the complex chemical reactions that go on in the cell (since enzymes control all of those reactions). This process of DNA-directed protein synthesis occurs in two stages: 1) transcription (messenger RNA synthesis: copying the genetic information from DNA to RNA) and 2) translation (polypeptide synthesis: using the genetic information in RNA to make a specific chain of amino acids). Beginning today, we will take a detailed look at transcription. Transcription is the process of making all of the cell's RNA molecules, not just those used in protein synthesis what are called the messenger RNAs (mRNAs). We will also take up what happens to those RNA molecules after they are made (RNA processing). Transcription overview video.
  • Transcription: Transcription is DNA-directed RNA synthesis (synthesis of RNA using DNA as the template).
    • In General: The sequence of a segment of a DNA molecule determines the sequence of an RNA molecule. RNA is very similar to DNA in structure, but 1) is usually shorter, 2) is usually single stranded (but may form double stranded loops call hairpin loops), 3) has ribose in place of 2'-deoxyribose, and 4) has uracil in place of thymine (uracil base pairs with adenine just like thymine does). Transcription is the process of making any RNA, whether it is mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), as well as other small RNAs (we will take up siRNAs and similar molecules later). (Nucleotide nomenclature
    • More Specifically: The enzyme(s) that synthesizes RNA is called RNA polymerase. RNA polymerase uses the nucleoside triphosphates (NTPs: ATP, GTP, CTP, UTP) and RNA polymerization occurs just like DNA polymerization does. That is, it begins with the 5' end and the new RNA molecule grows in the 5' to 3' direction (a nucleotide is added to the 3'-OH). RNA polymerase uses one strand of the DNA molecule as the template strand with the 4 bases of DNA (A, G, C, T) specifying which RNA nucleotides will be added (U is added when the template DNA nitrogen base is A). As with DNA replication, the newly-made RNA molecule is antiparallel to the template DNA. However, unlike DNA polymerase, RNA polymerase does not need a primer but can add start a new RNA molecule with a single NTP (therefore, the first nucleotide of an RNA molecule has 3 phosphates on its 5' end). Only one strand of the DNA double helix is used as a template. The strand that is copied is called the template strand and the strand not used is the non-template strand. Any RNA molecule made by transcription is called a transcript.
    • Transcription in Prokaryotes: The details of transcription were first elucidated in the bacterium E. coli.
      • E. coli's RNA Polymerase and Its Action: E. coli has only one RNA polymerase which makes all of the cell's RNA. The enzyme consists of 6 subunits (polypeptides)(α2, β, β', ω, σ). This make up the holoenzyme. However, the σ subunit is loosely attached and the enzyme without σ will still synthesize RNA, however not as efficiently and it will not begin at the right place. Therefore, σ is  needed for properly initiation of an RNA molecule (needed to start at the right spot on the DNA--called the promoter). This RNA polymerase without σ is called the core enzyme. RNA synthesis can be divided into three phases: initiation, elongation, and termination.
        • Initiation: The initiation of mRNA synthesis begins at a site on the DNA molecule called the promoter (RNA polymerase binds here). The promoter has two important regions (each about 6 nt long) at -10 and -35 (this means 10 and 35 bp upstream from--or before--the site where mRNA synthesis begins). σ binds to these two sites. These sites were discovered by the technique called protein footprinting (read and know about footprinting in your text). The entire RNA polymerase holoenzyme binds to a sequence that spans the 60 bases from -40 to +20. When the holoenzymes first binds to DNA, the DNA is still closed (base paired). Upon initiation, a 12-14 bp segment opens up (about -12 to +2) and RNA synthesis begins (the place where transcription begins is designated +1).
        • Elongation: RNA synthesis continues as the RNA polymerase "moves down" the DNA molecule. After about 10 nucleotides of RNA are synthesized, σ is released (it is not needed after initiation and can be used to start another transcript). As RNA polymerase "moves" down the DNA molecule, it "opens" the double helix ahead and closes it behind, always maintaining about 15 bp open. In the middle of this process, about 8-9 bases of the 3'-end of the mRNA are always base paired to the template DNA strand.
        • Termination: When the mRNA molecule is completed, termination releases the RNA from the DNA. This process occurs by one of two methods.

          • GC-Rich Sequence: In some cases, the end of the gene is marked by a sequence rich in Gs and Cs followed by a series of A bases (see figure). When this GC-rich region is transcribed, the RNA can base pair with itself forming a hairpin loop, while the RNA's U bases remain base paired with the DNA's As. The stronger bonding of the internal G-C pairs may disrupt the A-U pairs (A of template DNA, U of RNA) causing the mRNA to be released from the complex (this "pulls" the RNA off of the DNA).
          • ρ-Mediated Termination: In some cases, the ρ protein binds to extended mRNA and terminates transcription. (Recent results concerning ρ-mediated transcription termination is here. (Molecular Biology, 5th ed., Weaver. McGraw-Hill Publishers)
    • Eukaryotes: Eukaryotes have several RNA polymerases, each with specific tasks. They all have 9 subunits, 5 of which are very similar to the E. coli holoenzyme subunits. The 3-D structure of eukaryotic and E. coli RNA polymerase is also very similar.
      • RNA Polymerase II: This enzyme makes pre-messenger RNA. (We call it pre-mRNA because, as we will see later, it must be processed to become real mRNA.) So, this is the RNA polymerase that transcribes the genes that make proteins. Only RNA polymerase II has the CTD (C-terminus domain--see diagram under Initiation below). (RNA polymerase II also makes some of the small RNAs we will see later.)

        • Transcription Factors: Eukaryotic RNA polymerase is different from prokaryotic RNA polymerase because it will not synthesize RNA without a number of other proteins (factors) that are not in integral part of the enzyme (unlike σ which is an integral part of E. coli RNA polymerase). These factors for RNA polymerase II are of two types.
          • General Transcription Factors: These factors are necessary for transcription of any pre-mRNA to occur. (Some will be cover in more detail in the next topic.)
          • Gene-Specific Transcription Factors: These factors are necessary for the transcription of particular genes. We will take these up later in this course when we discuss the regulation of gene expression in eukaryotes.
        • Stages of Transcription: As in E. coli, transcription involves initiation, elongation, and termination.
          • Initiation: The promoter usually includes a sequence called the TATA box that is at about -30 to -25. (Some genes do not have a TATA box but have other sequences involved in initiation.) One of the general transcription factors is TFIID which binds to the promoter first and includes TBP (TATA-binding protein)(see figure). Then TFIIB binds which enables RNA polymerase binding followed by the binding of other general transcription factors. One of these is TFIIH which includes the enzyme helicase, which uncoils the DNA double helix.
          • Elongation: Elongation of the pre-mRNA continues with the aid of elongation factors.
          • Termination: Termination involves cleavage of the pre-mRNA and its polyadenylation, so it will be covered under "RNA Processing."
      • RNA Polymerase I and III: These RNA polymerases transcribe short RNAs. RNA polymerase I makes the larger rRNA molecules while RNA polymerase III makes the tRNAs and the smallest rRNA and some other small RNAs. (See "RNA Processing" for more details on these two enzymes.)
      • Other Polymerases: Chloroplasts and mitochondria have their own RNA polymerases.
  • RNA Processing: When transcription is finished, the newly made RNA molecules are altered, in some cases considerably. (Recent information!)
    • rRNA Processing: In eukaryotes, RNA polymerase I makes a large RNA molecule called pre-rRNA (45S) which is subsequently cut into three pieces yielding the 28S, 18S, and 5.8S rRNA molecules. The small 5.8S molecule hydrogen bonds to the end of the 28S molecule. (In E. coli, a similar process produces three E. coli rRNAs.)(Ribosomal subunits) The smallest eukaryote rRNA (5S) is made from a separate gene by RNA polymerase III. All eukaryote rRNA genes are tandemly repeated (up to several hundred times). (E. coli's rRNA genes are also repeated, but only about 3-10 times.) This repetition is presumably due to the fact that the cell must be able to make a lot of this transcript (rRNA) in order to make ribosomes. After rRNA synthesis, the molecules are chemically modified by methylation of some bases and of ribose. Also, uracil may be modified into an altered base. These rRNA modification occurs in the nucleolus (more later in the course on this structure and the assembly of ribosomes).

    • tRNA Processing: Eukaryotic tRNAs are made by RNA polymerase III. They are usually made as long precursor molecules, sometimes consisting of more than one tRNA. RNase P cuts the pre-tRNA near the 5' end and another enzyme cuts it near the 3' end (see 2010 article). RNase P is a complex of RNA and protein and the surprising discovery of Altman in 1983 was that it was the RNA that was acting as the catalyst (catalytic RNA, ribozyme). After the 3' end is cut, another enzyme adds a CCA trinucleotide to the 3' end (if the CCA is not already there). tRNAs undergo extensive base modification which will be important in translation. (Some tRNA have introns that must be removed, but this is done by "normal" protein enzymes, not catalytic RNAs -- see discussion on splicing.)

    • mRNA Processing: Unlike prokaryotic mRNAs, eukaryotic RNAs that code for protein are not ready to begin the translation process as soon as transcription ends (or in the middle of it, as in prokaryotes). That is, they are not yet  "real" mRNAs. These pre-mRNAs (sometimes called primary transcripts) are made by RNA polymerase II. This RNA polymerase has a unique domain called the C-terminus domain (CTD) where factors that process the pre-mRNA bind. The other eukaryotic RNA polymerases (I and III) do not have this CTD so they will not be process the same way pre-mRNA are. Pre-mRNA processing involves several events.
      • 7-Methylguanosine Capping: After about 20-30 nucleotides are synthesized, a GTP binds in reverse configuration to the 5' end of the pre-mRNA. This is then methylated. This methylguanosine cap stabilizes (it cannot be digested by exonucleases) the RNA and will be important in the translation process.
      • Polyadenylation: Another event is the enzymatic addition of about 200 adenine nucleotides to the 3' end of the pre-mRNA. The sequence AAUAAA occur about 10 nucleotides before a spot where there is a CA. A set of protein factors associated with RNA polymerase II's CTD cuts off the RNA on the 3' side of the CA then this same set of proteins adds the poly-A tail. The actual addition of the poly-A tail is by PAP (polyadenylate polymerase), which is one of these CTD-associated proteins. It uses ATP as the building blocks and build the poly-A tail one nucleotide at at time. This poly-A tail is needed for the mRNA's transport to the cytoplasm (as a specific protein binds to it causing its transport), for its stability, and for the translation most mRNAs. The polyadenylation/cleavage stimulates RNA polymerase II to slip off of the DNA template thus terminating transcription.
      • Splicing: A 1977 discovery rocked the scientific world. Up until that time, it was assumed that transcription and translation in prokaryotes and eukaryotes was essentially the same process. However, it was discovered that the pre-mRNA molecule of eukaryotes has several internal segment cut out and discarded. The first clues came from the fact that mRNA is much shorter than pre-mRNA. Furthermore, capping and polyadenylation occur before the RNA is shortened and these modifications remain in the finished mRNA. So, how does it get shorter if the ends are preserved? The answer came with the discovery of introns (mRNA-DNA hybridization experiments). Eukaryotic genes are transcribed into pre-mRNA and then certain segments (introns) are snipped out and discarded and the remaining segments (exons) are spliced together. The average human gene has about 8 introns which make up over 80% of the gene (average gene = 30 kb, average combined exon length = 2.5 kb).

        • The Process of Splicing: Splicing occurs in two steps as listed below. (When capping, polyadenylation, and splicing are finished, we can legitimately call this molecule mRNA. It will then be transported to the cytoplasm for translation, exiting through the nuclear pores.)
          • Lariat Formation: The pre-mRNA is cut at the 5' splice site (5' end of the intron) and simultaneously that newly-cut 5' end binds to the branch point adenine nucleotide (5' end to 2'-OH of the A nucleotide). This forms a lariat- (lasso-) shaped intermediate.
          • Splicing: The 3' splice site (3' end of the intron) is cut and simultaneously the two exons are joined. The lariat is then opened and degraded. These two steps are catalyzed by the spliceosome which includes proteins (in human there are probably over 170 proteins) and small nuclear RNAs (snRNAs, from 50 - 200 nt long, called U1, U2, U4, U5, U6). A spliceosome therefore is a small nuclear ribonucleoprotein (snRNP). Since U2 and U6 can catalyze splicing by themselves, the catalytic activity of the spliceosome is in the RNA (as with RNase P). A short sequence at the 5' end of the U1 snRNA is complementary to the 5' splice sites of the pre-mRNA. During splicing, U1 binds first to start the process. Another snRNA, U2, and proteins bind with U2 base pairing at the branch point. U6, U5, U4 and more proteins bind. U6 appears to associate with the 3' splice site and U2 associates with U6. There is evidence that U5 associates with exon sequences near both the 5' and 3' splice sites, possibly tethering the exons together until they are joined. (See this page for an overview of the roles of the snRNAs.)(Some self-splicing RNAs have been discovered in Tetrahymena.)
          • (Splicing is no longer a eukaryote-only process!)

      • RNA Editing: RNA bases may be changed after transcription, resulting in a change in the gene product (amino acid sequence). In one case in humans, apolipoprotein B mRNA can have a base changed by editing that results in the termination of translation (CAA ---> UAA, a nonsense codon). As a result, the 4536 amino acid-long protein found in liver is shortened to a 2152 amino acid-long protein in the intestine. Also, A ---> I changes are a common mode of RNA editing. (Serotonin receptor mRNA can be edited at 5 sites.)
        • Squid are hyper-editors when it comes to RNA (by Lisa D. Chong, Science Feb 27, 2015): During RNA editing, specific enzymes alter nucleotides in mRNA transcripts so that the resulting protein differs in amino acid sequence from what was encoded by the original DNA. Such RNA editing is a means to generate greater protein diversity; however, most organisms only use it sparingly. Alon et al. (eLife 4, e05198, 2015), however, now report an exception. They sequenced RNA and DNA from the squid nervous system and discovered that 60% of the transcripts exhibited RNA editing. Such "recoding" occurred largely in genes with cytoskeletal or neuronal functions and may be advantageous to organisms such as squid that must respond quickly and continually to environmental changes.
      • RNA Degradation: The expression of a gene is a function of the rate of production of its mRNA and the rate of degradation of that mRNA (among other things). Therefore, RNA degradation is an important process. Cells have a nonsense-mediated mRNA decay process which destroys mRNA that have a premature nonsense codon. "Old" mRNA are routinely degraded. The half life of prokaryotic mRNA is about 2-3 minutes versus 30 minutes to about 20 hours in eukaryotes. (mRNA degradation)(measuring RNA synthesis and degradation)

Nucleotide Nomenclature
Nitrogen Base Nucleoside (N Base+Sugar) Nucleotide (N Base+Sugar+PO4) Nucleotide used in RNA synthesis
adenine (A) adenosine adenylic acid
(adenosine monophosphate: AMP)
adenosine triphosphate (ATP)
guanine (G) guanosine guanylic acid
(guanosine monophosphate: GMP)
guanosine triphosphate (GTP)
cytosine (C) cytidine cytidylic acid
(cytidine monophosphate: CMP)
cytidine triphosphate (CTP)
uracil (U) uridine uridylic acid
(uridine monophosphate: UMP)
uridine triphosphate (UTP)
Nitrogen Base Nucleoside (N Base + Sugar) Nucleotide (N Base + Sugar + PO4) Nucleotide used in DNA synthesis
adenine (A) deoxyadenosine deoxyadenylic acid
(deoxyadenosine monophosphate: dAMP)
deoxyadenosine triphosphate (dATP)
guanine (G) deoxyguanosine deoxyguanylic acid
deoxyguanosine monophosphate: dGMP)
deoxyguanosine triphosphate (dGTP)
cytosine (C) deoxycytidine deoxycytidylic acid
deoxycytidine monophosphate: dCMP)
deoxycytidine triphosphate (dCTP)
thymine (T) deoxythymidine deoxythymidylic acid
deoxythymidine monophosphate: dTMP)
deoxythymidine triphosphate (dTTP)

Things I Learned at the Movies: It does not matter if you are heavily outnumbered in a fight involving martial arts--your enemies will wait patiently to attack you one by one by dancing around in a threatening manner until you have knocked out their predecessors.

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