Genetic Code & Transcription

Genetic Code and the Directional Flow of Genetic Info

• what does DNA serve as? what does this direct?

• what is the final product sometimes?

• what is the central dogma of molecular biology?

• DNA serves as a template for the synthesis of an RNA molecule, which then directs the synthesis of a protein product

• sometimes, the RNA itself is the final product

• the principle of directional information flow from DNA to RNA to protein is the central dogma of molecular biology

Transcription vs Translation

• Transcription: RNA synthesis using DNA as a template

• Translation: synthesis of protein using the information in the RNA

Mutations that alter sequences near the 5′ end of mRNA result in alterations near the corresponding protein’s N-terminal end, whereas mutations that alter the 3′ sequences of mRNA result in alterations in the protein’s C-terminal end. What do these findings imply?

A.The N- and C-terminal ends of proteins are susceptible to alteration.

B.The genetic code is a series of amino acids (the component parts of proteins).

C.The order of nucleotides from 5′ to 3′ in mRNA determines the order of amino acids from N- to
C-termini.

D.The genetic code is universal.

C.The order of nucleotides from 5′ to 3′ in mRNA determines the order of amino acids from N- to
C-termini.

Transcription and Translation Involve Many of the Same Components in Bacteria and Eukaryotes

• what is mRNA?

• rRNA?

• tRNA?

• Messenger RNA, mRNA, is RNA that is translated into protein

• Ribosomal RNA, rRNA, is an integral component of the ribosome

• Transfer RNA, tRNA, molecules serve as intermediaries, bringing amino acids to the ribosome

• The latter two function in translation

Transcription & Translation image

The Genetic Code

• what is it?

• what is a doublet and triplet code?

• The relationship between the DNA base sequence and the linear order of amino acids in the protein products is based on a set of rules known as the genetic code

1. There are four DNA bases and 20 amino acids

2. A doublet code, in which two bases specify a single amino acid, is inadequate because only 16 combinations are possible

3. A triplet code, in which combinations of three bases specify amino acids, would have 64 possible combinations, more than enough for all 20 amino acids

Supporting a Triplet Code using Frameshift Mutations

• what does inserting or deleting a nucleotide do?

• what are mutations that called this called?

• ex?

• Inserting or deleting a nucleotide (indel mutations) causes the rest of the sequence to be read out of phase—this is a shift in the reading frame

• Mutations that cause insertion or deletion of a nucleotide are thus called frameshift mutations

a) Proflavin is a mutagen that induces insertion or deletion of single nucleotides

b) experiments using Proflavin supported the idea of a triplet code

The interpretation of Revertible Mutations

• single, double, triple mutants?

• No revertant single mutants

• Revertant double mutants if the two mutations are opposite

• Revertant triple mutants only if all mutations are the same

The Genetic Code Is Degenerate and Nonoverlapping

• how many combinations of nucleotides are there, how many amino acids?

• what does this mean?

• There are 64 combinations of nucleotide triplets and only 20 amino acids

• This means the genetic code is a degenerate code, meaning that a particular amino acid can be specified by more than one triplet

• It is also nonoverlapping; the reading frame advances three nucleotides at a time

Nonoverlapping vs Overlapping Code

The Codon Dictionary Was Established Using Synthetic RNA Polymers and Triplets

• what are codons?

• what did homopolymer experiments show?

• RNA triplets, called codons, are read by the transcriptional machinery

• Further homopolymer experiments showed AAA codes for lysine, and CCC codes for proline

• As synthetic polymer technology progressed, production of all different codons independently led to the elucidation of the entire codon dictionary

Genetic code is universal for all organisms

Messenger RNA Guides the Synthesis of Polypeptide Chains

• how is mRNA transcribed?

• mRNA is transcribed from DNA similarly to how DNA is replicated, but with two differences

• In mRNA synthesis, only one DNA strand is copied, called the template strand; the other strand is called the coding strand because it is similar to the mRNA sequence

• In mRNA synthesis, a uracil base (U) is used instead of thymine

Transcription Involves Four Stages: Binding, Initiation, Elongation, and Termination

• what is the transcription unit?

• when does transcription begin? explain the 4 steps

• The DNA that gives rise to one RNA molecule is called the transcription unit

• Transcription begins when RNA polymerase binds to a promoter sequence (1), triggering local unwinding of the double helix

• RNA polymerase then initiates synthesis of RNA using one DNA strand as a template (2)

• After initiation, the RNA polymerase moves along the DNA template, unwinding the helix and elongating the RNA (3)

• Eventually, RNA polymerase dissociates from the DNA template, leading to termination of synthesis and release of the RNA molecule (4)

Transcription Image

• what determines where RNA synthesis will start?

• what does upstream and downstream mean?

•RNA polymerase binds to a DNA promoter site, a sequence of several dozen base pairs that determines where RNA synthesis will start

•The terms upstream and downstream refer to sequences located toward the 5′ or 3′ end of the transcription unit, respectively

Essential Sequences in a Typical Bacterial Promoter

• what is the transcription start site usually? AUTGC?

• what is about 10 bp upstream of the start site?

• 10 sequence? -35 sequence?

• The transcription start site is almost always a purine and usually an adenine

• About 10 bp upstream of the start site is the sequence TATAAT, called the –10 sequence or the Pribnow box

• At or near the –35 position is the sequence TTGACA, called the –35 sequence

Bacterial RNA Polymerase Structure and Initiation of Transcription IMAGE

Elongation of the RNA Chain

• which end is each new nucleotide added to?

• what is unwound and rewound?

• Chain elongation continues as RNA polymerase moves along the DNA molecule

• The RNA is elongated in the 5′ to 3′ direction, with each new nucleotide added to the 3′ end

• As the polymerase moves along the DNA strand, the double helix ahead of the polymerase is unwound, and the DNA behind it is rewound into a double helix

Elongation of RNA Chain image

Termination of RNA Synthesis

• what is the termination signal?

• what do many termination sequences contain?

• what do they form?

• Elongation of the RNA chain proceeds until the RNA polymerase copies a sequence called the termination signal

• Many termination sequences contain a short GC-rich sequence followed by several U’s

• The GC region in the RNA forms a hairpin loop pulling the RNA molecule away from the DNA

• Then the bonds between the U’s and the A’s of the template strand break, releasing the RNA

Transcription in Eukaryotic Cells Has Additional Complexity Compared with Prokaryotes

• what does eukaryotic transcription involve?

• what are the differences?

• Eukaryotic transcription involves the same four stages as prokaryotic, but there are several important differences

• Three different RNA polymerases transcribe one or more different classes of RNA

• Eukaryotic promoters are more varied than bacterial ones; some are even located downstream of the gene

Eukaryotic Transcription

• what are transcription factors?

• what type of interaction is in eukaryotic transcription?

• Eukaryotic transcription differs from that of prokaryotes

• RNA polymerases in eukaryotes require additional proteins called transcription factors, some of which must bind before the RNA polymerase can bind

• Protein-protein interactions play a prominent role in eukaryotic transcription

Eukaryotic Transcription (continued)

• what is more important than termination of transcription?

• what do newly forming RNA molecules undergo?

• Eukaryotic transcription differs from that of prokaryotes

• RNA cleavage is more important than termination of transcription in determining the 3′ end of the transcript

• Newly forming RNA molecules undergo RNA processing, chemical modification during and after transcription

RNA Polymerase I, II, and III Carry Out Transcription in the Eukaryotic Nucleus

• location?

• main products?

• a-Amanitin Sensitivity

• (Only for RNA Polymerase II and III)

• There are three RNA polymerases in the nucleus, designated RNA polymerases I, II, and III

• We will focus on just II and III

Three Classes of Promoters Are Found in Eukaryotic Nuclear Genes, One for Each Type of RNA Polymerase

• what is the core promoter?

• The core promoter is the smallest set of DNA sequences that initiates transcription

• We will compare the core promoter elements for the promoters used by RNA Polymerases II and III

The Promoter for RNA Polymerase II

• what are the 4 types of DNA sequences that are involved in the core promoter function?

• At least four types of DNA sequences are involved in core promoter function

1. A short initiator sequence surrounds the transcription start point

2.The TATA box, a consensus sequence of TATA followed by two to three A’s, is located about 25 nucleotides upstream of the start point

3.The TFIIB recognition element (BRE) is located slightly upstream of the TATA box

4.The downstream promoter element (DPE) is located about 30 nucleotides downstream from the start point

TATA-driven Promoters

TATA-driven Promoters:

Contain:

• TATA bos

• Inr

May or may not contain:

• BRE

No DPE Sequence

DPE-driven promoters

DPE-driven promoters

Contain:

• DPE sequence

• lnr

NO TATAbox

NO BRE

Promoters for RNA Polymerase III

• what location are the promoters at that are used by RNA polymerase III

• what boxes does tRNA have rRNA have?

• RNA polymerase III uses promoters that are entirely downstream of the start point

• In both 5S RNA and tRNA, the promoters are different, but both consensus sequences fall into two blocks of about 10 bp each

• tRNA has box A and box B; rRNA has box A and box C

• where does transcription start?

• what are consensus sequences transcribed into?

• Transcriptional start site is at upstream end of promoter

• Consensus sequences are all transcribed into RNA

Additional Control Elements

• how much transcription are core promoters able to drive?

• what improves promoters efficiency?

• what are proximal control elements?

• what are those farther away called?

• Core promoters are capable of driving only a basal (low) level of transcription

• Additional short sequences upstream (upstream control elements) improve the promoter’s efficiency

• Those within 100–200 nucleotides of the start point are called proximal control elements

• Those farther away are called enhancer elements or distal control elements

General Transcription Factors Are Involved in the Transcription of All Nuclear Genes

• what are general transcription factors required for?

• what factors do eukaryotes have?

• what does a large complex of proteins form?

• A general transcription factor is always required for RNA polymerase binding to promoters

• Eukaryotes have many such factors, called TFs, that bind the promoter in a defined order starting with TFIID

• Eventually, a large complex of proteins forms a preinitiation complex on the promoter

Initiation at a RNA Polymerase II Promoter

• what is essential for beginning the process?

• what does it do?

• what lead to the recruitment of RNA polymerase II?

• TFIID is essential for beginning the process

• TFIID recognizes and binds DNA because of its TATA-binding protein (TBP) subunit

• Other TFs follow TFIID, many binding to each other and not directly to DNA

• Leads to recruitment of RNA Polymerase II

Initiation at a RNA Polymerase II Promoter

• what activity does TFIIH have?

• what does this promote?

TFIIH has both helicase and kinase activity

•Local unwinding of DNA

•Phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II

Both promote the release of RNA Polymerase II from the initiation complex and the beginning of transcription

Termination of Eukaryotic RNA Synthesis

• what is termination governed by?

• for RNA polymerase III what do the termination signals include?

• what happens to the RNA polymerase II transcripts?

• where is the cleavage sit?

• Termination is governed by signals that differ for each type of RNA polymerase

• For RNA polymerase III, termination signals include a short run of U’s, and no protein factors are required for their recognition

• For RNA polymerase II, transcripts are cleaved at a specific site before transcription ceases

• The cleavage site is 10–35 nucleotides downstream of a AAUAAA sequence in the RNA

Termination of Transcription

• For RNA polymerase III, termination signals include a short run of U’s, and no protein factors are required for their recognition

• For RNA polymerase II, transcripts are cleaved at a specific site before transcription ceases

• The cleavage site is 10–35 nucleotides downstream of a AAUAAA sequence in the RNA

Cleavage location image

• RNA Polymerase II continues through the AAUAAA sequence

• The pre-mRNA is cleaved 10-35 nt after the AAUAAA and released

RNA Processing and Turnover

• what is the primary transcript?

• what must is undergo?

• A newly produced RNA molecule is called the primary transcript

• It must undergo RNA processing (chemical modification) before it can function in the cell

A. rRNA, tRNA, and mRNA

Transfer RNA Processing Involves Removal, Addition, and Chemical Modification of Nucleotides

• what do cells do?

• what does their secondary structure contain?

• what structure to tRNAs have?

• Cells synthesize several dozen kinds of tRNA molecules

• They fold into a secondary structure, most containing four hairpin loops; but some have a fifth region called a variable loop

• tRNAs have a cloverleaf structure and are synthesized as pre-tRNAs, followed by processing

Primary Transcript for yeast tyrosine tRNA vs Mature tRNA, secondary structure

Eukaryotic Transcripts

• what is hnRNA?

• how are Pre-mRNAs processed?

• what does the C- terminal domain of one of the subunits of RNA polymerase II act as?

• hnRNA is a mixture of mRNA molecules and their precursors, pre-mRNA

• Pre-mRNAs are processed by removal of sequences and addition of 5′ caps and 3′ tails

• The C-terminal domain of one of the subunits of RNA polymerase II acts as a platform for protein complexes involved in processing

5′ Caps and 3′ Poly(A) Tails

• what is the 5’ cap?

• how is it bound by the RNA molecule?

• Eukaryotic mRNAs have a modified nucleotide called the 5′ cap, and the 3′ ends have a long stretch of adenines called the poly(A) tail

• The 5′ cap is a guanosine that is methylated at position 7 of the purine ring

• It is bound to the RNA molecule by a 5′→5′ linkage rather than the usual 3′→5′ bond

5’ Cap

• what does it contribute to?

• what role does it play?

•The 5′ cap is a guanosine that is methylated at position 7 of the purine ring

•It is bound to the RNA molecule by a 5′→5′ linkage rather than the usual 3′→5′ bond

•The cap contributes to mRNA stability by protecting the RNA from nucleases

•The cap also plays a role in positioning the RNA on the ribosome for initiation of translation

5’ Cap Photo

The Poly(A) Tail

• how many nucleotides long is it?

• what is it added by?

• where is the signal for the addition of the poly(A) tail located? (sequence)

• function?

• what is it required for?

• The poly(A) tail ranges from 50 to 250 nucleotides long and is added by the enzyme poly(A) polymerase

• A signal for addition of the poly(A) tail, AAUAAA, is located just upstream of the polyadenylation site, and a GU- or U-rich element is located downstream of it

•The poly(A) tail protects the mRNA from nuclease attack; the length of the tail influences stability

•It is also required for export of the transcript to the cytoplasm

•It may also help ribosomes recognize and bind mRNAs

The Poly(A) Tail Image

Spliceosomes Remove Introns from Pre-mRNA

• what is RNA splicing?

• Sequences commonly found at the intron-exon boundaries likely determine by what?

• what does the 5’ end and start with and terminate with at the 3’ end?

• The process of removing introns and joining the exons is RNA splicing

• Sequences commonly found at the intron-exon boundaries likely determine the 5′ and 3′ splice sites

Analysis of base sequences of hundreds of different introns revealed that the 5′ end of an intron typically starts with GU and terminates with AG at the 3′ end

Primary Transcript (pre-mRNA)

The Splicesome

• what are spliceosomes?

• where do they assemble?

• what are snRNPs?

• Intron removal is catalyzed by large molecular complexes called spliceosomes, consisting of five types of RNA and many proteins

• Spliceosomes assemble on transcripts from a group of smaller RNA-protein complexes called snRNPs (small nuclear ribonuclearprotein complexes), each containing one or two snRNA molecules (small nuclear RNA)

Spliceosome Image

Splicesome Assembly

• what are they assembled by?

• what are the steps?

• Spliceosomes are assembled by sequential binding of snRNPs to pre-mRNA

• The first step is the binding of a snRNP called U1, which contains an RNA that can base-pair with the 5′ splice site

• A second snRNP called U2 binds to the branch-point sequence

Splicesome Assembly (continued)

• 4,5,6 step

• resulting structure?

• Finally a group of snRNPs (U4/U6 and U5) brings the ends of the intron together to form a mature spliceosome

• The pre-mRNA is cleaved at the 5′ splice site, which is joined to an adenine residue located at the branch-point sequence

• The resulting structure is called a lariat

Spliceosome Assembly (continued)

• what happens after the lariat is formed?

• what is an exon junction complex (EJC)? where is it deposited?

• After the lariat forms, the 3′ splice site is cleaved, and the two ends of the exon are joined together

• A multiprotein complex called an exon junction complex (EJC) is deposited near the boundary of each newly formed exon-exon junction

Alternative Splicing

• how is it possible? via what mechanisms?

• The presence of introns allows each gene’s pre-mRNA molecule to be spliced in multiple ways, leading to production of multiple protein products

• This alternative splicing is possible via mechanisms allowing certain splice sites to be activated or skipped

Alternative Splicing Image

C.Weaker H-bonding of A-U interactions compared to G-C interactions

The C-Terminal Domain of RNA Polymerase II Coordinates RNA Processing

• what occurs cotranscriptionally?

• what domain is responsible for this processing?

• what do repeats of a seven amino acid sequence of the CTD do?

•Many RNA processing events occur cotranscriptionally

•The long C-terminal domain (CTD) of RNA polymerase is responsible for this processing

•Many repeats of a seven-amino-acid sequence on the CTD bind enzymes needed for capping, splicing, and cleavage/polyadenlylation

Most mRNA Molecules Have a Relatively Short Life Span

• what do most mRNA molecules have?

• how are turnover rates measured?

• how long are the half-lives of mRNA molecules of eukaryotes?

• what about bacteria?

• Most mRNA molecules have a high turnover rate (rate at which molecules are degraded and replaced)

• It is measured in terms of half-life, the time required for 50% of the molecules to degrade

• mRNA molecules of eukaryotes have half-lives of several hours to a few days; in bacteria, the half-lives are usually only a few minutes

The Abundance of mRNA Allows Amplification of Genetic Information

• what provides the opportunity for amplification of genetic info?

• ex?

• mRNA can be synthesized again and again from a piece of template DNA, providing an opportunity for amplification of genetic information

• For example, the haploid genome of the silkworm has only one copy of the fibroin gene, but about 10⁴ copies of the mRNA are present in the cell at any given time