Chapter 12- Learning Objectives

Outline the central dogma of genetics (see Figure 12.1).

 The flow of genetic information is from DNA to RNA to polypeptide (protein)
 DNA Replication:
o Makes DNA copies that are transmitted from cell to cell and parent to
offspring
o Chromosomal DNA stores information in units called genes
 Transcription:
o Produces a RNA copy of a gene
 Messenger RNA is a temporary copy of a gene that contains
information to make a polypeptide
o A template strand provides a templae for ordering the sequence of
complementary nucleotides in an RNA transcript
 Template strand is always the same strand for any given gene
 The other strand is the coding strand which resembles the mRNA
coding strand
 Translation:
o Produces a polypeptide using the information in mRNA
 Polypeptide becomes a part of a functional protein that contributes
to an organism’s traits
Describe the general organization of a structural gene (see Figure 12.2).
 DNA:
o Regulatory sequences: binding of regulatory proteins to influence the rate
of transcription
o Promoter: site for RNA polymerase binding which signals the beginning of
transcription
o Terminator: signals the end of transcription
 mRNA:
o Ribosome-binding site: translation begins near this site in mRNA
 Eukaryotic ribosomes scan the mRNA for a start codon
o Start-codon specifies the first amino acid in a polypeptide sequence
o Codon: 3-nucleotide sequences within the mRNA that specify particular
amino acids
 The sequence of codons within m RNA determines the sequence of
amino acids within a polypeptide
o Stop-codon specifies the end of polypeptide synthesis
o **bacterial mRNA may be polycistronic which means is encodes two or
more polypeptides
Describe the three stages of transcription (see Figure 12.3).
1. Initiation: the initial binding of RNA polymerase to the promoter in order to
begin RNA synthesis
a. Promoter is a recognition site for transcription factors which enable RNA
polymerase to bind to the promoter
b. After binding, the RNA polymerase is denatured into a bubble (open
complex)
2. Elongation: synthesis of RNA transcript
a. RNA polymerase slides along the DNA in an open complex to synthesize
RNA
 3. Termination: a terminator is reached that causes RNA polymerase and RNA
transcript to dissociate from the DNA
Describe the features of bacterial promoters, but don’t memorize the actual sequences (see
Figures 12.4, 12.5).
 Promoters promote gene expression
 ***short sequences are critical for promoter recognition
 Transcriptional start site is the first base used as a template for transcription (+1)
o Bases preceding this site are numbered in a negative direction (no base is zero)
 Sequence elements at -35 and -10 sites play a key role in promoting transcription

Explain how bacterial RNA polymerase binds to a promoter and transcribes a gene (see Figures
12.7, 12.8).
1. RNA polymerase holoenzyme assembles into 6 subunits and loosely binds to
DNA
2. Holoenzyme then slides along DNA
3. When the holoenzyme encounters a promoter, a sigma factor recognizes the -35
and -10 sequences
4. A helix-turn-helix motif from the sigma factor tightly binds to the -35 and -10
sequence
5. Alpha helices from the sigma protein fit into the major groove of the DNA double
helix and form hydrogen bonds w/ bases
6. A closed complex is formed when the sigma factor binds to the promoter
7. Unwinding occurs at the TATAAT in -10 and an open complex is formed
8. A short strand of RNA is made and the sigma factor is released from the core
enzyme
9. The core enzyme slides down the DNA to synthesize a strand of RNA
10. RNA polymerase moves along the template in 3’ to 5’ and RNA is synthesized in
5’ to 3’ using nucleoside triphosphates
a. Pyrophosphate is released
b. U instead of T

Outline the two mechanisms of transcriptional termination in bacteria (see Figure 12.10, 12.11).
 RHO-DEPENDENT TERMINATION
o Rho-protein functions as helicase and separates RNA-DNA regions
o Rho-utilization site is synthesized by RNA
o Rho-protein binds to rut site and moves in the direction of RNA
polymerase
o At terminator sire, DNA encodes and RNA sequence containing several
GC base pairs that form a stem loop
o Stem loops causes RNA polymerase to pause
o RNA synthesis terminates several nucleotides beyond stem loop.
 RHO-INDEPENDENT TERMINATION
o When RNA polymerase reaches end of terminator, a uracil-rich sequence
is transcribed
o Stem loop forms upstream from open complex and causes RNA
polymerase to pause synthesis
 Pausing is stabilized by NusA
o During pause, RNA binds to DNA
o Transcript and RNA polymerase dissociate from DNA due to weak
hydrogen bonds
Compare and contrast the functions of the three eukaryotic RNA polymerases.
 RNA polymerase I:
o Transcribes all rRNA genes EXCEPT 5S rRNA
 RNA polymerase II:
o Transcribes all protein-encoding genes. (synthesis of all mRNAs)
o Transcribes genes for most snRNAs needed for splicing
o Transcribes several genes that produce non-coding RNAs
 RNA polymerase III:
o Transcribes all tRNAs and 5S rRNA
o Transcribes a few non-coding RNAs
 *** They are all structurally similar & composed of many subunits
o Two large catalytic Beta subunits

Describe the organization of protein-encoding genes in eukaryotes (see Figure 12.13).

 Start site occurs at Adenine; two pyrimidines (cytosine or thymine); and cytosine
is to the left of the adenine, and five pyrimidines are to the right
 TATA box 25bp from start site (not all protein-encoding genes have a TATA box)
 Regulatory elements (GC or CAAT boxes) are found in -50 to -100bp
 Single upstream regulatory element is involved in the binding of RNA polymerase
I to its promoter
 A and B boxes facilitate the binding of RNA polymerase III

Explain how the binding of general transcription factors and RNA polymerase is an assembly
process (see Figure 12.14).
 TFIID binds to TATA box
o TFIID is a complex of proteins that includes the TATA-binding protein
and several TBP-associated factors
 TFIIB binds to TFIID
o TFIIB promotes the binding of RNA polymerase II to the core promoter
 TFIIF binds to RNA polymerase II (by the promotion from TFIBB)
 TFIIE and TFIIH binds to RNA polymerase II to form a preinitiation/closed
complex
 TFIIH acts as a helicase to form an open complex.
 TFIIH phosphorylates the CTD of RNA polymerase II
 TFIIB-E-H are all released

Outline two possible mechanisms for transcriptional termination in eukaryotes (see Figure
12.15).
 ALLOSTERIC:
o RNA poly II becomes destabilized after it transcribes the polyA signal
sequence
o It eventually dissociates from DNA
 Destabilization may be caused by the release of proteins that
function as elongation factors or by the binding of proteins that
function as termination factors
 TORPEDO: RNA poly II is physically removed from DNA
o The region of RNA that is being transcribed downstream from the
polyadenylation signal sequence is cleaved by an exonuclease that
degrades the transcript in the 5’ to 3’ direction
 o When exonuclease catches up to RNA pol II, RNA poly II dissociates from
DNA

With regard to splicing, explain how pre-mRNA splicing occurs in eukaryotes (see Figures
12.19, 12.20).
Spliceosome subunit functions:
1. Bind to an intron sequence and precisely recognize the intron-exon boundaries
2. Hold the pre-mRNA in the correct configuration to ensure the splicing together of
exons
3. Catalyzes the chemical reactions that cause the intron to be removed and the exons
to be covalently linked
 U1 binds to 5’ splice site
 U2 binds to branch site
 U4/U6 and U5 trimer binds
o Intron loops out and exons are brought closer together
 5’ splice site is cut
 5’ end of intron is connected to the A in the branch site to form a lariat
 U1 and U4 are released
 3’ splice sit is cut
 Exon 1 is connected to exon 2
 Lariat intron is released with U2, U5, and U6.
 Intron is degraded
Explain how alternative splicing occurs (see Figures 12.21, 12.22).
 Constitutive exons: encode polypeptide segments of the alpha-tropomyosin protein
that are necessary for its general structure and function
 Alternative Exons: polypeptide sequences encoded by these may subtly change the
function of alpha tropomyosin to meet the needs of the cell type
 Repressors prevent the recognition of splice sites (skipping)
 Enhancers enhance the recognition of splice sites (addition)
Describe capping and tailing of mRNAs, but don’t memorize the chemistry of capping (see
Figures 12.23, 12.24).
 Capping: the covalent attachment of a 7-methylguanosine to the 5’ of mRNA
1. Occurs while pre-mRNA is made by RNA poly II (20-25bp)
2. Enzyme RNA 5’-triphosphatase removes one phosphate
3. Enzyme guanylyl transferase hydrolyzes guanosine triphosphate to attach a
guanosine monophosphate to the 5’ end.
4. Methyltransferase attaches a methyl group to a N at 7 in guanine
 Tailing: polyA tail is added by enzymes after the pre-mRNA has been completely
transcribed
1. Endonuclease cuts RNA 11-30 nucleotides downstream from AAUAA
polyadenylation sequence (making the RNA shorter at 3’ end)
2. Adenine-containing nucleotides are attached one at a time to the 3’ end by
enzyme polyA-polymerase