BSCI222: Principles of Genetics: Exam 3

DNA is…

Semi-conservative Replication

each of the original nucleotide strands remind intact, despite their no longer being combined in the same molecule, with the other strand being newly synthesized

Conservative Replication

the entire double-stranded DNA molecule serves as a template for a whole new molecule of DNA, and the original DNA molecule is fully conserved during replication

Dispersive Replication

both nucleotide strands break down (disperse) into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA molecules. Each resulting DNA molecule contains interspersed fragments of old and new DNA

What is a segment of DNA that undergoes replication called?

Replicon

What does each replicon contain?

Origin of Replication

How many origins of replication does bacteria have?

One

How many origins of replication does a eukaryote have?

Many

What happens during theta replication?

the two nucleotide strands of a circular DNA molecule unwind, creating a replication bubble. Within each replication bubble, DNA is normally synthesized on both strands and at the replication fork, producing two circular DNA molecules

What is a replication fork?

The point of unwinding, where the two strands separate from the double strand DNA helix

What does the unwinding of the double helix generate?

Replication bubble

Where does rolling circle replication take place?

viruses and F factor of E. Coli

Where does theta replication take place?

Circular DNA

What happens during rolling circle replication?

initiated by a break in one strand of circular DNA, which produces a 3’ OH group to which new nucleotides are added while the 5’ end of the broken strand is displaced from the circle

What happens in the Linear Eukaryotic DNA replication model?

Unwinding and replication take place on both templates at both ends of the replication bubble until adjacent replicons meet, resulting in two linear DNA molecules

What are the requirements of replication

• A template consisting of single stranded DNA

• Raw materials (substrates) are needed to be assembled into a new nucleotide strand

• Enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule

What direction is DNA synthesized in?

5’ → 3”

How does replication take place (due to antiparallel nature of DNA)?

continuously on one strand (the leading strand) and discontinuously on the other (the lagging strand).

What are Okazaki fragments?

The short lengths of DNA produced by discontinuous replication of the lagging strand

What does each active replication fork require?

• Helicase to unwind DNA

• Single stranded binding proteins to protect the single nucleotide strands and prevent secondary structures

• DNA gyrase to remove strain ahead of the replication fork

• Primase to synthesize primers

• DNA polymerase to synthesize the leading and lagging strands

What are the mechanisms that ensure the high rate of accuracy in replication?

precise nucleotide selection, proofreading, and mismatch repair

Homologous recombination

takes place through alignment of homologous DNA segments, breaks in nucleotide strands, and rejoining of the strands. requires a number of enzymes and proteins

Initiator protein

Binds to origin and separates strands of DNA to initiate replication

DNA helicase

Unwinds DNA at replication fork

Single strand binding proteins

Attach to single stranded DNA and prevent secondary structures from forming

DNA gyrase

Moves ahead of the replication fork, making and resealing breaks in the double-helical DNA to release the torque that builds up as a result of unwinding at the replication fork

DNA primase

Synthesizes a short RNA primer to provide a 3’ -OH group for the attachment of DNA nucleotides

DNA polymerase III

Elongates a new nucleotide strand from the 3’ -OH group provided by the primer

DNA polymerase I

Removes RNA primers and replaces them with DNA

DNA ligase

Joins Okazaki fragments by sealing breaks in the sugar-phosphate backbone of newly synthesized DNA

What are ribozymes?

catalytic RNA molecules that can cut out parts of their own sequences, connect some RNA molecules together, replicate others, and catalyze the formation of peptide bonds between amino acids

One difference in DNA and RNA replication

In replication, all of the nucleotides in DNA are copied, but in transcription, only parts are transcribed into RNA

What are the three major components required for transcription?

• A DNA template

• Raw materials needed to build a new RNA molecule

• The transcription apparatus, consisting of the proteins necessary for catalyzing the synthesis of RNA

What is the template strand?

The nucleotide strand used for transcription (leading strand)

What is the name of the strand that is not transcriped?

The lagging strand (non-template strand)

What are three critical regions in a transcription unit?

• Promoter

• an RNA coding region

• Terminator

What is the promoter?

DNA sequence that the transcription apparatus recognizes and binds to

What is the RNA coding region?

a sequence of DNA nucleotides that is copied into an RNA molecule

What is the terminator?

A sequence of nucleotides that signals where transcription is going to end

How many RNA polymerases does bacteria have?

Only one

How many RNA polymerases does a eukaryote have?

multiple types

What is RNA polymerase?

consists of a core enzyme, which catalyzes the addition of nucleotides to an RNA molecule and other subunits. The sigma factor controls the binding of the core enzyme to the promoter

What happens during initiation of transcription?

in which the transcription apparatus assembles on the promoter and begins the synthesis of RNA

What happens during elongation of transcription?

in which DNA is threaded through RNA polymerase and the polymerase unwinds the DNA and adds new nucleotides, one at a time, to the 3’ end of the growing RNA strand

What happens during termination of transcription?

the recognition of the end of the transcription unit and the separation of the RNA molecule from the DNA template

What are the two types of terminators in bacteria cells?

Rho dependent and Rho independent terminators

What is a rho dependent terminator?

It is able to cause the termination of transcription only in the presence of an ancillary protein called the rho factor

What is a rho independent terminator?

it is able to cause the end of transcription in the absence of the rho factor

Ribosomal RNA

In prokaryotic and eukaryotic cells, in the cytoplasm, functions as the structural and functional component of the ribosome

Messenger RNA

In both types of cells, nucleus and cytoplasm, and carries the genetic code for proteins

Transfer RNA

in both prokaryotic and eukaryotic cells, located in the cytoplasm, and helps to incorporate amino acids into the polypeptide chain

Pre-mRNA

Eukaryotic cells only, in the nucleus, and is a precursor to mRNA

Small Nuclear RNA

Eularyotic cells only, in the nucleus, and aids in the processing of pre-mRNA

Small Nucleolar RNA

only in eukaryotes, in the nucleus, and processes and assembles rRNA

Micro RNA

Only in eukaryotes, in both the nucleus and cytoplasm, and inhibits translation of mRNA

Small interfering RNA

In eukaryotes, in both the cytoplasm and nucleus, and triggers degradation of RNA molecules

Piwi interacting RNA

only in eukaryotic cells, in both the nucleus and cytoplasm, and suppresses transcription of transposable elements in reproductive cells

Long Noncoding RNa

only in eukaryotic cells, both in the nucleus and cytoplasm, and performs a variety of functions

CRISPR RNA

only in prokaryotic cells and assists the destruction of foreign DNA

RNA polymerase 1

Present in all eukaryotes and transcribes large rRNAs

RNA polymerase 2

Present in all eukaryotes and transcribes pre-mRNAs, snoRNAs, some miRNAs, and some snRNAs

RNA Polymerase 3

Present in all eukaryotes and transcribes tRNAs, small rRNAs, some miRNAs, some snRNAs

RNA polymerase 4

only in plants and transcribes siRNAs that silence transposons

RNA polymerase 5

only in plants and transcribes siRNAs that affect chromatin

What are introns?

noncoding regions called intervening sequences

Where are introns mainly found?

they are common in eukaryotic genes but very rare in bacterial genes

What are exons?

They are RNA coding regions

What three primary regions does any mRNA molecule have?

• 5’ untranslated region

• Protein coding region

• 3’ untranslated region

What is the 5’ untranslated region?

a sequence of nucleotides at the 5’ end of the mRNA that does not encode any amino acids or proteins

What is the protein coding region?

A region comprised of only the codons that specify the amino acid sequence of the protein (begins with a start codon and end with a stop codon)

What is the 3’ untranslated region?

A sequence of nucleotides at the 3’ end of the mRNA that is not translated into amino acids

What structure removes introns from pre-mRNA?

Spliceosome

What is the spliceosome composed of?

Several small nuclear RNAs and proteins

What is RNA editing?

when the coding sequence of mRNA is altered after transcription, resulting in a translated protein with a different amino acid sequence that differs from that encoded by the gene

How many subunits does a functional ribosome consist of?

Two: Large ribosomal subunit and small ribosomal subunit

Theoretical Argument for a Triplet Code

Scientists, notably George Gamow and Francis Crick, theorized that a code of at least three nucleotides (a triplet) was necessary to specify 20 amino acids. A single nucleotide would only code for four amino acids (4^1), and a two-nucleotide code for sixteen (4^2), insufficient for all twenty. A triplet code (4^3, or 64 combinations) provided more than enough capacity. This work established that the code words, or codons, must be three bases long

The "Poly-U" Experiment

In 1961, Marshall Nirenberg and Heinrich Matthaei conducted a pioneering experiment using a cell-free bacterial extract system. They added a synthetic messenger RNA (mRNA) made entirely of uracil (poly-U) to this system, along with radioactively labeled amino acids, and observed what protein was produced. The result was a protein chain composed solely of the amino acid phenylalanine. This experiment cracked the first "word" of the genetic code, revealing that the codon UUU specifies phenylalanine

Frameshift experiments

The Frob experiment by Francis Crick, Sydney Brenner, and colleagues used induced mutations (insertions and deletions) in a gene. They found that single or double base pair insertions or deletions caused a frameshift mutation that rendered the protein non-functional. However, inserting or deleting three base pairs often restored most of the gene's function. This demonstrated experimentally that the genetic code is read in a non-overlapping sequence of three nucleotides, confirming the triplet nature of the code and that translation begins from a fixed starting point.

The Filter-Binding Assay

While the poly-U experiment identified some codons, deciphering the rest using long, synthetic, random co-polymers was difficult due to ambiguity. In 1964, Nirenberg and Philip Leder developed a new method using short, synthetic, three-nucleotide mRNA fragments (specific codons). They showed that these fragments would bind to a ribosome and, in turn, bind the appropriate transfer RNA (tRNA) charged with a specific radioactively labeled amino acid. By testing all 64 possible triplets, they could quickly and unambiguously determine which amino acid each codon specified, allowing the entire genetic code to be mapped by 1966.

One gene, one enzyme hypothesis

genes function by encoding enzymes, and each gene encodes a separate enzyme

One gene, one polypeptide hypothesis

some proteins are composed of more than one polypeptide chain and that different polypeptide chains are encoded by separate genes

Synonymous codons

codons that specify the same amino acid

Wobble

where different codons may pair with the same anticodon, allows some nonstandard pairing of bases at the third position of the codon

Isoaccepting tRNAs

different tRNAs may accept the same amino acid but have different anticodons

What four steps does protein synthesis consist of?

• tRNA charging

• Initiation

• Elongation

• Termination

What is tRNA charging?

where tRNAs bind to amino acids

what happens during initiation of protein synthesis?

components necessary for translation are assembled at the ribosome

What happens during elongation of protein synthesis

amino acids are joined to the growing polypeptide chain

what happens during termination of protein synthesis

protein synthesis stops at the termination codon and translation components are released from the ribosome

What is translocation?

movement of the ribosome from the 5’ → 3’ direction, along mRNA to the next codon

What are mRNA surveillance mechanisms?

mechanisms that eliminates mRNAs with errors that may create problems in translation

What are post-translational modifications?

• Nonsense mediated mRNA decay

• nonstop mRNA decay

• no go decay

What happens during nonsense mediated mRNA decay?

rapid elimination of mRNAs with premature termination codons

What happens during nonstop mRNA decay?

rapid degradation of abnormal mRNA

What happens during no go decay?

helps remove stalled ribosomes resulting from secondary structures in the mRNA, chemical damage, premature stop codons, and ribosomal defects

What are structural genes?

genes that encode proteins that are used in metabolism or biosynthesis or that play a structural role in the cell

What are regulatory genes?

genes that encode RNA or proteins that interact with other DNA sequences and affect the transcription or translation of those sequences

What are constitutive genes?

not regulated genes that are expressed continually

What are operons?

groups of functionally related structural genes and the sequences that control their transcription

Transcription Negative control

When a repressor protein binds to DNA and inhibits transcription